Mutated parvovirus structural proteins as vaccines

ABSTRACT

The present invention is related to a method for identifying a parvovirus mutated structural protein capable of specifically binding to a binder for an antigen, a parvovirus mutated structural protein which comprises at least one B-cell epitope heterologous to the parvovirus, a multimeric structure comprising the protein, a nucleic acid encoding the protein, a virus or cell comprising the protein, a method of preparing the protein, a medicament comprising the protein, nucleic acid or multimeric structure and its use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 12/601,639, which has a 371 (c) date of Mar. 23, 2010, which is the U.S. National Stage of International Application No. PCT/EP2008/004366, filed Jun. 2, 2008, which claims the benefit of U.S. Patent Application No. 60/932,446, filed May 31, 2007, and European Patent Application No. 07013264.2, filed Jul. 6, 2007.

BACKGROUND OF THE INVENTION

The present invention is related to a method for identifying a parvovirus mutated structural protein capable of specifically binding to a binder for an antigen, a parvovirus mutated structural protein which comprises at least one B-cell epitope heterologous to the parvovirus, a multimeric structure comprising the protein, a nucleic acid encoding the protein, a virus or cell comprising the protein, a method of preparing the protein, a medicament comprising the protein, nucleic acid or multimeric structure and its use.

Monoclonal antibody therapies have been one of the most successful therapy forms of new drug developments over the last couple of years in therapeutic fields such as oncology, autoimmune and inflammatory diseases. In monoclonal antibody therapies patients are injected with a specific monoclonal antibody that recognizes the antigen involved in the disease. Antibodies recognize their antigen with the variable domain of the antibody which is also referred to as the idiotype of the antibody.

However, monoclonal antibody therapies also have certain drawbacks. It can be observed that, if the concentration of a specific antibody with one particular idiotype is too high, the patient's immune system develops an antibody response against the idiotype of the therapeutic monoclonal antibody and thereby limits its efficacy. This kind of antibody that recognizes an antibody's idiotype is referred to as an anti-idiotype antibody. In addition, antibodies to monoclonal therapeutic antibodies directed against other parts of the monoclonals often limit efficacy of a passive antibody therapy. Therefore, many of the monoclonal antibody drugs need to be used in combination with the traditional immunosuppression regiments, increasing the overall treatment costs. Furthermore, active suppression of the patient's immune system is detrimental especially, if an intact immune system is required to control the stage of disease such as for oncological indications.

As being a passive vaccination against the target antigen the monoclonal antibody has to be injected frequently depending on the half life of the antibody within the serum of the patient. Therefore, such treatments are expensive and inconvenient for the patients.

An alternative for such monoclonal antibody therapies already exists exemplified by a number of clinical developments using anti-idiotype antibodies as drugs. Such anti-idiotypic antibody therapies are based on the fact (see above) that the patient's immune system can induce an antibody response against the idiotype of an antibody. If one uses a monoclonal antibody expressing a functional imitation of a target epitope (paratope or mimotope) as an idiotype, the patient's immune system will generate a polyclonal antibody response wherein a subset of these antibodies is able to cross-react with the target epitope in the patient. Such antibody expressing a paratope is referred to an anti-idiotypic antibody (based on Jerne's network model of idiotypic relationships (Jerne, 1974, Jerne et al., 1982). Thus, selective immunization with an anti-idiotypic antibody can induce a specific immune response directed against the original antigen (Varela and Coutinho, 1991, Jefferis, 1993, Chatterjee et al., 1994).

Therefore, a vaccination with such an anti-idiotypic antibody actively induces a polyclonal antibody response. As a consequence such anti-idiotypic antibody vaccines have a number of advantages over a passive immunization by a standard monoclonal antibody. There is no antibody response towards the anti-idiotypic antibody that limits its efficacy as exactly this immune response is used as the therapeutic principle. Therefore, it is also not necessary to combine the antibody treatment with an immunosuppression regimen. And further, due to the fact that the anti-idiotypic treatment is an active immunization, the drug only has to be injected from time to time to boost the antibody response generated by the patient himself maintaining a continuous titer of specific antibodies. Additionally, anti-idiotype antibodies induce a polyclonal antibody response against the target antigen that hampers the potential mechanism for resistance to the treatment of e.g. in tumor cells.

However, anti-idiotypic antibody therapies face major disadvantages. The titers of the induced polyclonal antibody response obtained by the vaccination with anti-idiotypic antibodies are often not high enough to establish a beneficial treatment. This is due to the lack of a strong antigen as a vaccine, since antibodies per definition are not very immunogenic. Furthermore, it is difficult to generate specific anti-idiotype vaccines because of this lack of immunogenicity and technical difficulties to identify anti-idiotypic antibodies.

A series of publications describes that an antigen placed in the context of an ordered surface of a viral particle—here a papilloma virus particle—can induce a B cell response that even can abrogate B cell tolerance to such antigen by direct crosslinking the respective B-cell receptor. Bovine papilloma virus-like particles (VLPs) conjugated to an Aβ peptide through biotin were used to generate an immune response against the self antigen Aβ (Li et al., 2004). Further, this group used bovine papilloma virus-like particles having the murine chemokine receptor mCCR5 inserted into an immunodominant site of the viral L1 protein to immunize mice leading to sera with high anti-CCR5 antibody titers despite the fact that CCR5 is a selfantigen. Further, a macaque L1-CCR5 fusion protein was used to immunize pig tail macaques. 4 of the 5 test animals produced CCR5 specific antibodies. In a further approach TNF-α was joined to VLPs by way of a biotin-streptavidin interaction (Chackerian et al., 2001). These VLPs were successful in generating an auto-antibody response in mice, whereas these antibodies bound native TNF-α (U.S. Pat. No. 6,719,978).

Therefore, papilloma VLPs have been shown to be a suitable backbone for the presentation of antigens to the immune system in order to generate strong B cell responses, probably because of their dense, ordered and closely packed array of vaccination epitopes. Due do their exceptionally strong B cell induction papilloma VLPs can be especially useful to overcome B cell tolerance to self antigens.

However, it is questionable if epitopes linked by biotin or inserted by an educated guess can possibly induce the generation of auto-antibodies for a wide range of tolerogens, as advantageous epitopes for vaccination may be three-dimensional and inserted epitopes may refold due to the specific environment of the insertion site. This is especially true for small antigens or individual epitopes, where influences of the viral capsid backbone are more relevant than in case of larger insertions.

Therefore, the problem of the instant invention was to find alternative or even superior methods to identify drug candidates useful as vaccines for the treatment of diseases, especially accessible to antibody therapies that avoid one or more of the above mentioned disadvantages (BPV based VLPs with conjugated or manually inserted tolerogen-derived epitopes).

The problem is solved by a screening method for identifying a parvovirus mutated structural protein capable of specifically binding to a binder for an antigen, the method comprising the steps of (a) providing a library of parvovirus virions expressing at least one mutated parvovirus structural protein, (b) providing a binder for an antigen, (c) selecting at least one parvovirus virion specifically binding to the binder, and (d) identifying (i) the parvovirus mutated structural protein or a mutated part thereof, or (ii) the gene or a mutated part thereof encoding the parvovirus mutated structural protein of the parvovirus virion selected in step c).

Parvovirures, especially Adeno-associated virus type 2, are well known in the art as viral vectors for gene therapy (Muzyczka, 1992). Further, the AAV2 structural proteins have been genetically modified to change the cellular tropism of AAV2 and thereby direct the virus to cells or tissues that are under normal conditions not infected by the wild-type AAV2. The first successful retargeting of AAV2 was published by Girod A. et al. (Girod et al., 1999), (WO 99/67393). The authors identified insertion sites for AAV that can be modified e.g. by insertion of short peptide sequences without destroying the capability of the structural proteins to assemble into virions. The insertion of a peptide sequence of choice that is displayed on the surface of the virion then leads to an altered cell tropism that has successfully been tested in vivo (White et al., 2004). The technology has been further developed to be used to reduce the antigenicity of AAV to escape from the immune system of patients that have neutralizing antibodies against AAV (Huttner et al., 2003); (WO 01/05990) and to modify the AAV virion's chromatographic properties to enable the efficient manufacture of AAV vectors for gene therapy (WO 01/05991). This work has been confirmed and further insertion sites have been identified (Shi et al., 2001), especially tables 1-5, page 1708 “Identification of optimal sites for heterologous ligand insertion”; (Shi and Bartlett, 2003), US 2002/0192823; (Wu et al., 2000)).

To improve the technology of retargeting AAV to desired cells or tissues, libraries of mutated structural proteins of AAV have been constructed and successfully used for the selection of AAV clones with altered cell tropism (Perabo et al., 2003, Lieber, 2003, Muller et al., 2003, WO 03/054197).

Parvovirus structural proteins have been known in the past to form virus-like particles that can be used for vaccination purposes. A vaccine containing hybrid recombinant parvovirus-like particles of pig parvovirus (PPV) and canine parvovirus (CPV) containing a CD8+ epitope from the lymphocytic choriomenigitis virus (LCMV) nucleoprotein protected mice against lethal infection with LCMV (Casal, 1999). The same was shown for PPV and CPV virus-like particles (VLPs) containing the C3:T epitope from poliovirus (Casal, 1999). Also B19 structural proteins have been applied in epitope delivery for vaccination purposes. VP-2 capsid proteins of human parvovirus B19 VLPs were used to display linear epitopes of human herpes simplex virus type 1 and mouse hepatitis virus A59 (Brown et al., 1994), U.S. Pat. No. 6,719,978).

However, these attempts have been used only for fairly large pathogenic epitopes and not with tolerogens or small antigens or even individual epitopes, where B cell tolerance has to be broken to have a beneficial effect for the patient.

Screening methods using parvovirus libraries have been previously described in WO 03/054197. Disclosed therein are screening methods to identify parvoviruses with an altered cell tropism. The authors further disclose an immunoselection step using antibodies such as patient sera to remove immunogenic parvoviruses from the pool of viruses (negative selection). However, a selection of a parvovirus virions specifically binding to the binder, e.g. for a virion binding to a therapeutic antibody, was not disclosed, being a positive selection.

Medicaments according to the present invention have numerous advantages over the prior art. The immune system of a mammal is specialized to generate strong antibody responses against viral capsid proteins due to the co-evolution of mammals and their immune system on one hand and viruses on the other hand. Strong antibody responses means titers of 1000 to 100.000 measured in a standard ELISA. Virus-like particles are highly immunogenic due to resemblance of a virus and thereby efficient uptake of such particles by antigen-presenting cells. The size of the virion, the density and symmetric order of B-cell epitopes and the optimal distance of about 50 to 100 Å between any two B-cell epitopes plays a major role regarding very strong T-cell independent B-cell responses mediated by direct cross-linking of the respective B-cell receptor breaking even B-cell tolerance against self-antigens or tolerogens (Szomolanyi-Tsuda and Welsh, 1998, Szomolanyi-Tsuda et al., 1998, Szomolanyi-Tsuda et al., 2000, Szomolanyi-Tsuda et al., 2001, Zinkernagel, 2002, Bachmann et al., 1993).

Taken together, such medicaments are capable of inducing a polyclonal immune response against certain B-cell epitopes that leads to an active immune response resulting in long lasting antibody titers. The multimeric structure of the virion contains a large number of densely packed identical epitopes directly cross-linking the respective receptor on B-cells and, thereby, inducing a T-cell independent B-cell response. The particulate structure of the medicament further supports the immune response via efficient uptake by antigen-presenting cells which activate T-cells finally triggering IgG class switch and hypermutation of activated B-cells, leading to the persistent release of high-affinity IgG antibodies and differentiation of B-cells into memory cells. Using the methods of the current invention such medicaments can easily be screened and produced.

The following definitions explain how the defined terms are to be interpreted in the context of the products, methods and uses of the present invention:

A “structural protein” means a protein that is part of the capsid of the virus. For parvoviruses the structural proteins are generally referred to as VP-1, VP-2 and/or VP-3.

A “mutated structural protein” means a structural protein that has at least one mutation in comparison to the respective structural protein of the wild-type virus.

A “parvovirus” means a member of the family of Parvoviridae containing several genera divided between 2 subfamilies Parvovirinae (Parvovirus, Erythrovirus, Dependovirus, Amdovirus and Bocavirus) and Densovirinae (Densovirus, Iteravirus, Brevidensovirus, Pefudensovirus and Contravirus) (Fields: Virology, fourth edition 2001, Volume 2, chapters 69 and 70, Lippincott Williams Wilkins, Philadelphia; http://virus.stanford.edu/parvo/parvovirus.html http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fs_parvo.htm#SubFamily1). Preferred parvoviruses are members of the genus Parvovirus, such as AAV1, AAV2, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV10, AAV11, AAV12, bovine AAV (b-AAV), canine AAV (CAAV), canine parvovirus (CPV), mouse parvovirus, minute virus of mice (MVM), B19, H1, avian AAV (AAAV), feline panleukopenia virus (FPV) and goose parvovirus (GPV).

Preferred parvoviruses are adeno-associated virus (AAV), Bovine AAV (b-AAV), canine AAV (CAAV), canine parvovirus (CPV), minute virus of mice (MVM), B19, H1, AAAV, feline panleukopenia virus (FPV) and goose parvovirus (GPV). Especially preferred are AAV1, AAV2, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV10, AAV11 or AAV12, especially AAV2.

The term “binder” refers to a molecule that specifically binds to its respective binding partner. Commonly used binders are antibodies, especially monoclonal antibodies, antibody derivatives such as single chain antibodies or antibody fragments. In principle all classes of antibodies can be used, preferred are IgG antibodies. Fragments or multimers of antibodies can equally be used. Commonly used fragments are single chain antibodies, Fab- or (Fab)₂-fragments. Examples of other suitable binders are protein scaffolds such as anticalins or lipocalins (Nygren and Skerra, 2004), receptors or parts thereof (e.g. soluble T-cell receptors), ankyrine, microbodies or aptamers.

The term “specifically binds” means that two molecules A and B, preferably proteins, bind to each other thereby generating complex AB with an affinity (K_(D)=k_(off)/k_(on)) of at least K_(D)=1×10⁻⁵ mol/l, preferably 1×10⁻⁷ mol/l, more preferably 1×10⁻⁸ mol/l, especially 1×10⁻⁹ mol/l.

The term “antigen” in the context of the products, methods and uses of the present invention refers to any target antigen against which an immune reaction should be induced. Such target antigens are usually antigens that are susceptible to the humoral immune response. They are usually proteins that may be posttranslationally modified, as for example glycosylated proteins. Preferred antigens are serum proteins, proteins that can be found at least under certain conditions (e.g. in a disease state) in the blood (e.g. CETP, IL-6, IL-17, TNF-α), and membrane proteins, especially receptor proteins (e.g. CD20, acetylcholine receptors, IL13R, EGFR). Especially preferred antigens are IgE, tumor-antigens (e.g. Melan A, high molecular weight melanoma associated antigen (HMW MAA), CA125, IL13R, Her2/NEU, L1 cell adhesion molecule), VEGF, EGFR, CD20, IL-9, IL-13, CETP (cholesterol ester transfer protein), TNF-family members (e.g. TNF-α), interleukins (IL-6, IL-17) or misfolded proteins leading to a protein aggregation and, therefore, causing conformational diseases (for an overview see Uversky et al., 2006), e.g. β-amyloid). Excluded from the above definition of “antigen” are parvovirus antigens, i.e. antigens inherent the unmutated parvovirus itself, e.g. derived from B19 (Klenerman et al., 2002).

“Heterologous” in the context of the present invention means a peptide sequence, e.g. an epitope that is not present on the parvovirus wild-type viral capsid and/or structural protein.

A “tolerogen” is a self-antigen that is—in its natural environment—accessible to the humoral immune system. It may be either secreted or otherwise released from a living cell or associated to the outer surface of or integrated into the cellular membrane. Generally speaking tolerogens do—under normal circumstances in contrast to e.g. autoimmune diseases—not evoke a specific immune response due to tolerance against the antigen which results from a previous exposure to the same antigen. Tolerance can occur due to central tolerance or peripheral tolerance. Central tolerance refers to tolerogens which corresponding antigens have been exposed to T cells in the thymus leading to elimination of the specific T cells. Peripheral tolerance occurs when antigens/epitopes/mimotopes/paratopes are presented to T cells without appropriate additional stimuli, commonly provided by inflammation leading to anergy. Still, it has been observed that tolerogens can induce to some extent regulatory B-cell responses (Vogel et al., 2004).

In one preferred embodiment this invention relates to tolerogens due to peripheral tolerance, preferably tolerogens derived from tumor antigens/epitopes/mimo-topes/paratopes. Tolerogens encompassed by this invention include peptides, nucleic acids, carbohydrates, and lipids, preferably peptides.

Preferred tolerogens are antigens on the surface of a cell, especially tumor cells, e.g. receptors, especially growth factor receptors, tumor antigens, viral receptors, CD20, acetylcholine receptors, interleukin receptors. Further preferred tolerogens can be blood proteins such as interleukins, IgE, cytokines, immunoglobulins, complement factors, CETP and VEGF.

A “tolerogen-derived epitope” of a specific tolerogen in the context of the products, methods and uses of the present invention refers to a B-cell epitope that

i) is identical to a B-cell epitope of the tolerogen, ii) a derivative (e.g. a mutant) of a B-cell epitope of the tolerogen that crossreacts with an antibody that binds the B-cell epitope of the tolerogen, iii) a mimotope of a B-cell epitope of the tolerogen, and/or iv) a paratope of a B-cell epitope of a tolerogen.

The length of a tolerogen-derived epitope is typically 4-30, preferably 5-20 and most preferably 5-15 amino acids.

The derivative of a B-cell epitope of a tolerogen may be generated by one or more amino acid substitutions, preferably one or more conservative amino acid substitutions, i.e. substitutions that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc. Further, derivatives may be obtained by one or more single amino acid deletion(s) and/or insertion(s).

“Crossreaction” or “crossreact” of B-cell epitopes with a specific monoclonal antibody means according to this invention that the affinity (K_(D)) of the epitopes with the antibody are within two magnitudes, preferably within one magnitude when comparing the B-cell epitope to its derivative.

Tolerogen-derived epitopes within the multimeric structure comprising parvovirus mutated structural proteins according to this invention are identical, resemble or mimic antigen stretches of a tolerogen that are—in their natural environment—accessible to the immune system, e.g. epitopes of membrane protein located in the extracellular part, serum proteins, immunoglobulins, plaque proteins. Such antigen stretches are preferably located on the surface of such protein within the body of a mammal, preferably a human.

A “mimotope” is a non-linear structural epitope composed of several amino acids derived from different regions of the linear sequence of the structural protein located in close neighborhood due to the overall tertiary structure of the capsid that is specifically bound by an antibody, or a linear epitope mimicking a discontiuous epitope of the structural protein.

A “paratope” is the antigen binding site that is specifically bound by an antibody.

The mimotope or paratope in the context of the present invention might consist of (parts of) the inserted peptide sequence alone or might be composed of inserted peptide and parvovirus core particle amino acid residues.

An “insertion” of (an) amino acid(s) is generally speaking an insertion of at least one heterologous amino acid into the sequence of—for this invention—a parvovirus structural protein. ‘Heterologous’ in this context means heterologous as compared to the virus, from which the parvovirus structural protein is derived from. The inserted amino acids can simply be inserted between two given amino acids of the parvovirus structural protein. An insertion of amino acids can also go along with a deletion of given amino acids of the parvovirus structural protein at the site of insertion, leading to a complete substitution (e.g. 10 given amino acids are substituted by 10 or more inserted amino acids) or partial substitution (e.g. 10 given amino acids are substituted by 8 inserted amino acids) of amino acids of the parvovirus structural protein.

The invention relates to a method for identifying a parvovirus mutated structural protein capable of specifically binding to a binder for an antigen, the method comprising the steps of (a) providing a library of parvovirus virions expressing at least one mutated parvovirus structural protein, (b) providing a binder for an antigen, (c) selecting at least one parvovirus virion specifically binding to the binder, and (d) identifying (i) the parvovirus mutated structural protein or a mutated part thereof, or (ii) the gene or a mutated part thereof encoding the parvovirus mutated structural protein, of the parvovirus virion selected in step (c). The identified gene or mutated part thereof can then be expressed in a cell to obtain the parvovirus mutated structural protein or mutated part thereof.

For identification the at least one gene or the mutated part thereof encoding the parvovirus mutated structural protein may be transferred into a cell, and a cell producing the parvovirus mutated structural protein capable of binding to the binder can be identified. The gene or the mutated part thereof encoding the parvovirus mutated structural protein can be cloned by transducing the gene into a cell and a cellular clone producing the parvovirus mutated structural protein capable of binding to the binder may be identified. Additionally or alternatively, the gene encoding the parvovirus mutated structural protein may be sequenced comprising the individual steps of obtaining bound virions, optionally amplifying the DNA contained within the virions, and sequencing. Sequencing can be performed by standard methods, e.g. after PCR-amplification of at least the part of the parvoviral structural protein that contains the insert. Amplification products can be cloned into a plasmid, and the plasmids can be transformed into bacteria. Single clones can be sequenced and this sequence information can then be used to generate AAV particles of clonal origin.

In case of AAV the identified capsid sequences can be cloned into a standard AAV helper plasmid or in a plasmid containing the full AAV genome. For example, the 587 insertion site of AAV2 is flanked by NotI/AscI restriction sites which can be used for subcloning of the identified peptide-coding sequences into different VP expression vectors. Alternatively, a large part of VP3 can be subcloned by BsiWI and a second restriction enzyme cutting the vector backbone 3′ of the cap ORF (e.g. XmaI in pUC19).

In a preferred embodiment the at least one parvovirus virion selected in step c) of the method of the invention is amplified by viral replication and subsequent packaging in a production cell under suitable conditions, wherein at least steps b) to c) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, preferably 1, 2, 3, 4 or 5 times. Optionally, a step for coupling genotype and phenotype of a selected mixture of parvovirus virions can be performed as it is described below for the coupling of a whole library.

The general principle of the identification of such a parvoviral mutated structural protein is that selections are done using a library of viruses displaying a random peptide on the capsid surface. Virus capsid mutants which are able to bind the used binder, e.g. antibody, are selected, can be amplified and are re-used for a new selection round. After each selection round the selected sequences can be analyzed. A selected parvoviral mutated structural protein clone and/or its respective nucleic acid sequence is then used to generate a medicament, i.e. a vaccine.

Preferred binders for the carrying out the identification step of the present invention are Fab or (Fab)₂ fragments. If whole IgG antibodies are used, the coupling of the antibody to a support such as a culture plate takes place at random so that the library will be exposed to different parts of the IgG antibody (e.g. the desired idiotype, or the large Fc part which is an undesired event). Therefore, AAV particles which do not only bind the antibody idiotype but which also can bind other parts of the IgG antibodies will be finally isolated. This can be avoided by using only Fab or (Fab)₂ fragments. If those fragments are commercially not available for a specific monoclonal antibody, they can be generated from whole IgG antibodies by commercial kits.

There are also alternatives how binding can be performed. In one embodiment the selecting step is performed using a binder that is immobilized on a carrier, preferably directly or via a linker. Such linker can again be linked through a second site of the binder (the first site is the site that is used for screening the library) to a support or surface, e.g. of 12-well plate, e.g. using antibodies as binders through an Fc-specific linker such as protein A or G, sepharose, Fc-specific antibodies or -fragments. The binding of the binder through a linker to the surface has the advantage that the binder is bound in a directed fashion that can avoid unspecific binding of parvovirus mutated structural proteins. By this means, mainly the idiotype of the antibody will be exposed to the particles of the library.

Supports or surfaces used for the method of the invention can either be the surface of Petri dish, 12-well plate or alike, but also suitable chromatography material such CNBr-activated Sepharose. In the latter case a batch method using the chromatography material in suspension can be used.

Alternatively, the selection step is performed using a binder in suspension. Here, binder and parvovirus mutated structural proteins are capable of forming complexes in suspension which reflects the situation in vivo best. Such complexes can be precipitated using standard immune precipitation with Fc-specific antibodies or anti antibody affinity chromatography. Further, binders can be captured by Sepharose A/G columns. Instead of standard immune precipitation magnetic beads coupled to the selection antibody or to any binder which binds to the modified parvoviral particle can be used to isolate the desired modifications out of the library pool.

Specificity of the selection step in either way can be enhanced by addition of at least one washing step to remove unbound or weakly bound parvovirus mutated structural proteins. Suitable washing conditions are e.g. high salt concentrations or use of detergents such as Tween.

Additionally, selected parvovirus virion is further selected for non-binding to a second binder. Such binder might for example be derived from antibodies neutralizing the respective core particle. Another class of binder would be antibodies induced by the same parvovirus virion without inserted peptide sequences. By this means of selection, undesired immunodominant epitopes might be excluded.

Transduction of the gene into a cell is preferably carried out under conditions, where the uptake of the DNA is independent of an infection pathway but occurs through unspecific uptake (e.g. pinocytosis or phagocytosis) further described in more detail below.

Alternatively or additionally, the selection step can be carried out on cells expressing a specific receptor for a binder of choice which is used for selecting the desired parvoviral variant. E.g. cells can be used which express the FcγRI receptor which is specific for any binder comprising an Fc-part of an IgG antibody. For this example, such FcγRI expressing cells can be transduced with a library pool of parvoviruses. First, a negative selection can be performed to avoid unspecific selection of parvoviral candidates which by themselves are able to transduce cells independently from an interaction of a binder with FcγRI. Therefore, FcγRI-expressing cells are incubated with the library pool. The supernatant (pool of parvoviruses which is not able to transduce the cells) is collected and subsequently incubated with the binder of choice (e.g. selection antibody) to perform the positive selection. In the positive selection parvoviruses decorated with the binder will be able to transduce FcγRI expressing cells through attachment of the binder to FcγRI on the surface of the cells. The transduced cells can subsequently be used to amplify the particles.

The specific affinity or avidity of the selected parvovirus mutated structural proteins for the binder can be further enhanced by the additional steps of (e) randomizing the gene encoding the parvovirus mutated structural protein, (f) packaging the randomized genes into a further library of parvoviruses and (g) repeating the steps (a) to (d) of the above selection method.

In a preferred embodiment the parvovirus mutated structural protein further comprises at least one random mutation compared to the respective parvovirus wild type structural protein e.g. with its mutated amino acid residue either directly or indirectly contributing to the overall avidity or affinity of the respective virus particle to the binder of an antigen due to the formation of structural epitopes, mimotopes or paratopes.

Further mutation of the capsid protein might be adequate to e.g. i) introduce additional or even identical B-cell epitopes of the same target antigen, and/or ii) B-cell epitopes of one or more further target protein(s) (multi-target vaccine), T-helper 1 (T_(H1)) epitope(s) to further promote the desired T_(H1) immune response, peptide sequence(s) to target antigen-presenting cells, or to obtain capsid mutants with reduced immunogenicity of the core particle. The latter might be one possibility to setup an efficient prime/boost regimen.

In a further preferred embodiment the further mutation might be adequate to introduce at least one cytotoxic T-cell epitope (CTL epitope). For both infectious diseases and cancer it is most useful to combine both humoral and cellular immune responses to fight these diseases. The multimeric structures according to this invention are in principle capable of pseudo-infecting cells. Accordingly these structures—like viruses—are able to enter cells, are processed to peptides, the peptides are loaded onto MHC class I and II molecules and finally presented to CD8- or CD4-positive T cells. The T-cells become stimulated after specific recognition of such processed peptide presented by MHC class I or II molecules. As a consequence of such stimulation CD8 cells may differentiate into cytotoxic T cells and then cause a cellular immune response. CD4 cells may develop into T helper cells which stimulate B cells to provide a humoral immune response or CD8-positive T cells to provide a cytotoxic immune response, which may themselves induce lysis of infected cells and other cells carrying and presenting the same peptide. Suitable CTL epitopes are known in the art for various cancer antigens or viral antigens, or they can be predicted from given antigen sequences using for example the peptide prediction program by Parker under http://www-bimas.cit.nih.gov/molbio/hla_bind/ {Parker, 1994 #865}. Proposed CTL epitopes can be validated according to the methods as exemplified for HPV-epitopes in U.S. Pat. No. 6,838,084, examples 2-8 (herein incorporated by reference). As processing of CTL epitopes occurs within the cell it is not necessary that such CTL epitopes are located on the surface or are present in a specific conformation.

It is a preferred embodiment of the present invention that an identical B-cell epitope is inserted at two insertion sites, especially in I-587 and I-453, if it is key to have a large number of identical peptides being optimally presented on the surface of a capsid, especially in the case if direct B-cell receptor (BCR) crosslinking is required for T-cell independent priming of B-cells and breaking of tolerance against self-antigens. A higher density of B-cell epitopes increases the likelihood of optimal peptide-specific BCR crosslinking which requires a defined distance between BCRs (e.g. about 5-10 nm), and therefore, respective B-cell epitopes being presented on a parvovirus capsid. As shown in this invention (FIG. 19), identical insertions of—in this case a β-amyloid epitope—into parvovirus capsids at two (or more) different sites at a time can lead to a higher affinity of the capsid to an antibody specifically recognizing the inserted epitope, here the β-amyloid epitope at insertion sites I-453 and I-587. Consequently, in this case it is preferred that the inserted peptide is a B-cell epitope, more preferred a tolerogen-derived epitope. Therefore, it is an especially preferred embodiment of this invention that an identical peptide is inserted at I-453 and I-587 and that this peptide is a B-cell epitope, most preferred a tolerogen-derived epitope.

Further preferred double insertion variants are all possible combinations of I-261, I-453, I-534, I-570, I-573 and I-587, preferably I-261 in combination with I-587 and I-261 in combination with I-453.

Moreover, a larger number of inserted B-cell epitopes decreases the probability for undesired immune reactions against the parvovirus backbone due to i) masking of natural parvovirus B-cell epi/mimotopes and/or ii) slight structural capsid changes rendering these natural B-cell epi/mimotopes less immunogenic. Accordingly, parvovirus structural proteins comprising at least three insertions are especially preferred.

In a preferred embodiment genotype and phenotype of each virion particle of the library is coupled. This means that the genomic mutant of the virion is identical to the phenotypic mutant of the same virion or, in other words, that each structurally modified virus codes for its structural protein mutant.

In contrast to a bacterial transformation, where only one bacteriophage is taken up by one bacterial cell, using transfection methods for eukaryotic cells many DNA copies (up to 1×10⁶) can be taken up per cell (Dean et al., 2005). Therefore, in the case of an AAV library one cell can replicate thousands of AAV genomes at the same time where each may express a different mutated structural protein with a different peptide sequence inserted into VP-1, VP-2, and/or VP-3 of AAV. At least some of these structural proteins can assemble a complete viral capsid (consisting of 5 VP-1, 5 VP-2 and 50 VP-3 proteins) encapsidating only one of the thousands of AAV genomes present in the cell. In case of a geno-/phenotypically coupled library at least 10%, preferably more than 25%, especially more than 50% of the resulting AAV particles have an encapsulated genome which codes for at least 25%, preferably more than 50%, especially more than 80% of the 60 VP proteins of which its capsid is composed. As a consequence, if an uncoupled library was used for a first screening against a target antibody, the chance that screened particles contained the genome coding for this specific peptide sequence might be very low.

In general, geno-/phenotypically coupled virion particles/libraries are obtained when introducing one single copy of the virus genome into each virion production cell entering the cell nucleus. This cell will only produce capsid protein variants encoded of exactly the introduced genome which is replicated and afterwards packaged into the mutant virion particle. Different experimental settings can ensure this:

To obtain a geno-/phenotypically coupled library of parvovirus virions a library of parvovirus virions is produced by transfecting a plasmid library into production cells under suitable conditions whereas a low copy number of viral genomes equal to or less than 100 genomes per cell is used, preferably equal to or less than 10 genomes, more preferably equal to or less than one genome per cell, resulting in geno-/phenotypically coupled virions/library. The overall transfection efficacy will be finally decisive for the ideal number of virus genomes per cell to be transfected.

The required amount of virus plasmid can be quantified, if e.g. autonomous replicating plasmids with similar size as the virus genome encoding a reporter gene such as GFP are used as a model system. Autonomous replicating plasmids are e.g. systems comprising SV40 origin of replication and large T antigen or the EBV (ebstein barr virus) P1 origin and EBNA. Increasing amounts of the self-replicating reporter gene plasmid are cotransfected with carrier DNA such as empty plasmid DNA (e.g. pUC derivates) keeping the amount of total DNA constant. In theory, each cell transfected with the reporter gene plasmid will, due to its self-replication, express sufficient amounts of reporter protein to be detected. At some ratio of reporter gene vector to carrier DNA, a further increase of reporter gene plasmid will lead to a corresponding increase in the number of transfected cells. By this means, the ideal amount of self-replicating reporter gene plasmid can be determined, reflecting the ideal amount of vector genomes.

Similarly, another read-out system for detection of successfully transfected cells are methods such as in-situ PCR to detect the transfected plasmid genome on a single cell level.

Alternatively, the geno-/phenotypically coupled library of parvovirus virions can be produced by transducing a (non- or partially coupled) virion library into production cells under suitable conditions at a ratio of genomes per cell of 5 to 5,000, preferably 10 to 1,000, more preferably 50 to 300, especially approximately 100, and selecting transduction conditions to be independent from infection pathways, particularly through unspecific uptake through pinocytosis and/or phagocytosis, resulting in geno-/phenotypically coupled virions/library. As it is known that a peptide insertion into the I-587 site of AAV2 frequently destroys (dependent of the sequence of the inserted peptide) the heparin binding motif required for efficient infection of HSPG-receptor containing cells such as HeLa or 293 cells, simple infection methods could bias the screening method and lead only to mutants that still can enter HeLa cells specifically through the respective receptor, in case of AAV2 through heparan sulfate proteoglycan (HSPG). Therefore, an unspecific uptake of the virus particle by the production cell is advantageous. Such unspecific uptake can be achieved by seeding production cells on immobilized parvovirus virions. A preferred embodiment relates therefore to a method, whereas the transduction of the parvovirus virion library is performed using production cells seeded on immobilized parvovirus virions. For this method, the virions are directly coated to a support/surface, e.g. a tissue culture plate. Alternatively, first a capsid specific antibody (in case of AAV2 for example A20) is coated to the support/surface and second the capsids are bound to the coated antibodies. The advantage in the latter case is that the antibody/virus particle complex, respectively the virus particle itself is more efficiently detached from the support/surface and thereby internalized by the cell. Importantly, introduction of foreign peptide sequences into I-587 of AAV2 does not destroy the affinity of A20 to the respective mutant particle as the epitopes of A20 are hardly, if at all, affected by the peptide insertion. The cells, e.g. HeLa cells, are finally seeded on the bound capsids. It is expected that this procedure leads to an uptake of the virus, e.g. AAV, by the cell independent of the natural infectious pathway, presumably by pinocytosis and/or phagocytosis.

In a further preferred embodiment a geno-/phenotypically coupled library of parvovirus virions can be obtained by a method where selected virions are specifically taken up by production cells. In this case the library of parvovirus virions is produced by transducing the library into production cells under suitable conditions at a ratio of genomes per cell of 10 to 10,000, preferably 50 to 5,000, more preferably 100 to 3,000, especially approximately 1,000, wherein transduction conditions are selected to be dependent on infection pathways, particularly through specific receptor binding, resulting in geno-/phenotypically coupled virions/library. In order to achieve such receptor-specific uptake the virions of the library are preferably not immobilized but added to the cells in suspension, whereas both cells and virions can be in suspension or cells are immobilized and virions are added in suspension. Therefore, the transfection of the cells is basically dependent on the virus's infection pathway. In this context it is conceivable to transduce FcγRI expressing cells as described above but incubating the selected pool with A20 antibody-decorated AAV particles (instead of incubating the pool with a binder).

Dependence on infection pathways means that virions are taken up by the cells e.g. through receptor-specific uptake, e.g. for AAV2 heparin sulfate proteoglycan (HSPG)-specific uptake (e.g. for virion libraries where natural infection pathways are not blocked or destroyed by the inserted random peptide sequences). For AAV2 particles with peptide sequences being inserted into I-587, infection of cells will work as long as the capsid contains sufficient HSPG binding motifs or binding motifs for secondary receptors expressed on the cell line used for the coupling step. An AAV capsid consists of 60 capsid proteins each containing the I-587 insertion site. Therefore, mosaic capsid virions containing a given percentage of wild-type sequence capsid proteins will still be able to infect cells via HSPG or secondary receptors. Alternatively, virions with peptide insertions partially restoring the affinity to HSPG or secondary receptors will be able to infect cells such as HeLa or 293. Especially peptide sequences containing basic amino acid residues such as lysine or arginine at the correct position have been shown to restore the natural HSPG infection pathway of I-587 AAV capsid mutants. Given the frequency of basic amino acid residues in a 7mer random sequence and given the fact that an AAV capsid consists of 60 capsid proteins, many if not most of the virions of a non-coupled I-587 AAV2 library consisting of particles with a mosaic capsid will still infect cells to a certain degree via HSPG receptor-mediated uptake.

To keep biodiversity of the library during the coupling step (either by transfection of virus genomes or by cell transduction with virion particles by either means, uptake or infection), always an at least 10-fold, preferably 100-fold, especially 500-fold excess of genomic particles compared to the multiplicity of parvoviral mutants should be transduced in order to ensure that each virus variant is amplified. To further ensure that each virus is coupled in the resulting library an at least 2-fold, preferably at least 5-fold excess of cells is to be used compared to total number of genomic particles.

Geno-/phenotype coupling is desired as the genetic information of the packed DNA can easily be used to obtain the sequence of those particles having high affinity or avidity to the respective antigen binder. It is an object of the invention to use for the identification of a parvovirus mutated structural protein such geno-/phenotypically coupled libraries with a coupling of at least 5%, preferably of at least 25% and more preferably of at least 50%, especially at least 90%.

In a preferred embodiment the library has a multiplicity of parvoviral mutants of greater than 10⁵, preferably greater than 10⁶, especially greater than 10⁷. Multiplicity means according to this invention the number of different virions or viral genomes within the library. In principal it is advantageous to use a library of high multiplicity as the likelihood to identify a suitable or even ideal clone increases with the multiplicity of the library.

The multiplicity of the library is generated by insertion of a nucleic acid insert into the coding region of the gene encoding a parvoviral structural protein leading to an amino acids insertion at a particular position within the parvoviral structural protein. It is preferred according to this invention that the insertion(s) is inserted into one or more positions selected from the group consisting of I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-453, I-459, I-471, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713 and I-716, more preferably I-261, I-453, I-534, I-570, I-573 and I-587, especially I-587.

The used nomenclature I-### refers to the insertion site with ### naming the amino acid number relative to the VP1 protein of AAV2, however meaning that the insertion may be located directly N- or C-terminal, preferably directly C-terminal of one amino acid in the sequence of 5 amino acids N- or C-terminal of the given amino acid, preferably 3, more preferably 2, especially 1 amino acid(s) N- or C-terminal of the given amino acid. For parvoviruses other than AAV2 the corresponding insertion sites can be identified by performing an amino acid alignment or by comparison of the capsid structures, if available. Such alignment has been performed for the parvoviruses AAV1, AAV-6, AAV2, AAV-3b, AAV-7, AAV-8, AAV10, AAV-4, AAV11, b-AAV, AAV-5, GPV, B19, MVM, FPV and CPV (see FIG. 1).

For example the insertion site I-587 corresponds to an insertion before and/or after one of the following amino acids indicated by emphasis

SEQ ID NO: 1: FQSSS TDPAT of AAV1, SEQ ID NO: 2: LQRGN₅₈₇  RQAAT of AAV2, SEQ ID NO: 3: LQSSN TAPTT of AAV-3b, SEQ ID NO: 4: LQSSS TDPAT of AAV-6, SEQ ID NO: 5: LQAAN TAAQT of AAV-7, SEQ ID NO: 6: LQQQN TAPQI of AAV-8, SEQ ID NO: 7: LQQAN TGPIV of AAV10, SEQ ID NO: 8: NQNAT TAPIT of AAV11 and SEQ ID NO: 9: NQSST TAPAT of AAV-5.

Further, the insertion site I-453 corresponds to an insertion directly N- or C-terminal of the following ten amino acids each, preferably directly C-terminal of the amino acid indicated by emphasis

SEQ ID NO: 10: QNQSG SAQNK of AAV1, SEQ ID NO: 11: NTPSG ₄₅₃ TTTQS of AAV2, SEQ ID NO: 12: GTTSG TTNQS of AAV-3b, SEQ ID NO: 13: QNQSG SAQNK of AAV-6, SEQ ID NO: 14: SNPGG TAGNR of AAV-7, SEQ ID NO: 15: QTTGG TANTQ of AAV-8, SEQ ID NO: 16: QSTGG TQGTQ of AAV10, SEQ ID NO: 17: SGETL NQGNA of AAV11 and SEQ ID NO: 18: FVSTN NTGGV of AAV-5.

Relating to the AAV2 sequence insertion sites for AAV and other parvoviruses encompassed by this invention are listed in Table 1.

TABLE 1 Insertion sites for parvoviruses Insertion corresp. site amino acid/sequence of AAV2 References I-1 M₁ M₁ AADGY SEQ ID NO: 19 (Wu et al., 2000) I-34 P₃₄ PPPKP₃₄ AERHK SEQ ID NO: 20 (Wu et al., 2000) I-138 T₁₃₈ EPVKT₁₃₈ APGKK SEQ ID NO: 21 (Wu et al., 2000, Warrington et al., 2004, Lux et al., 2005) I-139 A₁₃₉ PVKTA₁₃₉ PGKKR SEQ ID NO: 22 (Shi et al., 2001, Shi and Bartlett, 2003, Arnold et al., 2006) I-161 K₁₆₁ SGTGK₁₆₁ AGQQP SEQ ID NO: 23 (Shi et al., 2001, Arnold et al., 2006) I-261 S₂₆₁ YKQIS₂₆₁ SQSGA SEQ ID NO: 24 (Girod et al., 1999) I-266 A₂₆₆ SQSGA₂₆₆ SNDNH SEQ ID NO: 25 (Wu et al., 2000) I-381 N₃₈₁ YLTLN₃₈₁ NGSQA SEQ ID NO: 26 (Girod et al., 1999) I-453 G₄₅₃ NTPSG₄₅₃ TTTQS SEQ ID NO: 11 data of this invention I-447 R₄₄₇ YYLSR₄₄₇ TNTPS SEQ ID NO: 27 (Girod et al., 1999, Wu et al., 2000) I-448 T₄₄₈ YLSRT₄₄₈ NTPSG SEQ ID NO: 28 (Grifman et al., 2001) I-459 R₄₅₉ TTQSR₄₅₉ LQFSQ SEQ ID NO: 29 (Shi et al., 2001, Arnold et al., 2006) I-471 R₄₇₁ ASDIR₄₇₁ DQSRN SEQ ID NO: 30 (Asokan and Samulski, 2006, Moskalenko et al., 2000) I-534 F₅₃₄ EEKFF₅₃₄ PQSGV SEQ ID NO: 31 (Girod et al., 1999) I-570 P₅₇₀ RTTNP₅₇₀  VATEQ SEQ ID NO: 202 data of this invention for Δ566-575 I-573 T₅₇₃ NPVAT₅₇₃ EQYGS SEQ ID NO: 32 (Girod et al., 1999) I-584 Q₅₈₄ STNLQ₅₈₄ RGNRQ SEQ ID NO: 33 (Shi et al., 2001, Shi and Bartlett, 2003) I-587 N₅₈₇ LQRGN₅₈₇ RQAAT SEQ ID NO: 2 (Girod et al., 1999, Shi et al., 2001, Maheshri et al., 2006, Ried et al., 2002, Grifman et al., 2001, Nicklin et al., 2001, Arnold et al., 2006) I-588 R₅₈₈ QRGNR₅₈₈ QAATA SEQ ID NO: 34 (Shi and Bartlett, 2003) I-591 A₅₉₁ NRQAA₅₉₁ TADVN SEQ ID NO: 35 (Wu et al., 2000) I-657 P₆₅₇ VPANP₆₅₇ STTFS SEQ ID NO: 36 I-664 A₆₆₄ TFSAA₆₆₄ KFASF SEQ ID NO: 37 (Wu et al., 2000) I-713 T₇₁₃ NVDFT₇₁₃ VDTNG SEQ ID NO: 38 I-716 T₇₁₆ FTVDT₇₁₆ NGVYS SEQ ID NO: 39 (Maheshri et al., 2006)

Amino acid 138 is the N-terminus of VP-2. Preferred embodiments are VP-2 structural proteins with an N-terminal fusion to one of the amino acids within the stretch T₁₃₈ APGKKR (SEQ ID NO: 40) of AAV2 or the corresponding amino acids of other parvoviruses.

I-570 is especially suitable as an insertion site that goes along with a deletion of given amino acids of the parvovirus structural protein at the site of insertion, leading to a complete substitution. In this case the amino acids RTTNPVATEQ can be substituted by an epi- or mimotope.

Further, the inserted nucleic acid sequence may be inserted at any site corresponding to the first amino-terminal amino acids 1 to 50 of VP-1.

Insertions have been successfully made into AAV-serotypes other than AAV2.

TABLE 2 Insertions into AAV-serotypes other than AAV2 Ins. site/ amino acid AAV relative to serotype Sequence AAV2 References AAV1 FQSSS₅₈₈ TDPAT SEQ ID NO: 1 I-587 N₅₈₇ own data AAV1 SSSTD₅₉₀ PATGD SEQ ID NO: 41 I-589 Q₅₈₉ (Arnold et al., 2006, Stachler and Bartlett, 2006) AAV-3 NNLQS₅₈₆-SNTAP SEQ ID NO: 42 I-585 R₅₈₅ (Arnold et al., 2006) AAV-4 GGDQS₅₈₄-NSNLP SEQ ID NO: 43 I-585 (Arnold et al., 2006) AAV-5 TNNQS₅₇₅-STTAP SEQ ID NO: 44 I-585 (Arnold et al., 2006)

The most preferred insertion sites are:

i) I-587 as various insertions have been made in the amino acid stretch around N₅₈₇ (LQRGN₅₈₇ RQAAT, SEQ ID NO: 2) of AAV2. Within this stretch insertions of various peptides were made C-terminal of amino acids Q₅₈₄, N₅₅₇, R₅₈₈ and A₅₉₁ in AAV2 (Table 1) and C-terminal of amino acids of other AAV-serotypes corresponding to R₅₈₅ and Q₅₈₉ of AAV2 (Table 2). ii) I-453 as according to this invention epitopes have been successfully inserted C-terminal of G₄₅₃ in AAV2. iii) FQSSS₅₈₈ TDPAT (SEQ ID NO: 1) or SSSTD₅₉₀ PATGD (SEQ ID NO: 41) of AAV1. iv) I-261 as according to this invention epitopes have been successfully inserted C-terminal of S₂₆₁ in AAV2. v) I-534 as according to this invention epitopes have been successfully inserted C-terminal of F₅₃₄ in AAV2. vi) I-570 as according to this invention epitopes have been successfully inserted C-terminal of P ₅₇₀ in AAV2. vii) I-573 as according to this invention epitopes have been successfully inserted C-terminal of T₅₇₃ in AAV2.

Corresponding amino acids for all insertion sites specified herein for parvoviruses disclosed herein can be retrieved from the alignment in FIG. 1, for those parvoviruses not listed herein an alignment under standard parameters as used herein can be formed with the provided amino acid sequence of such parvovirus and the corresponding amino acids can be retrieved from such alignment.

The amino acid numbers are given relative to the VP-1 amino acid sequence. However, insertions into the structural gene encoding the structural protein may generally also lead to mutated VP-2 and optionally VP-3 proteins comprising an insertion at a site which is corresponding to the VP-1 insertion as VP-2 and VP-3 are generally expressed from the identical structural gene using downstream located start codons for the start of translation leading to—compared to VP-1—N-terminally truncated structural proteins. A schematic organization of the cap gene of AAV2 is provided in FIG. 2. Therefore, the present inventions encompasses structural genes of parvoviruses with corresponding insertions in the VP-1, VP-2 and/or VP-3 proteins. For example for AAV2, insertions into the cap gene between the codons coding for amino acids 1 and 138 lead to an insertion only in VP-1, insertions between codons coding for amino acids 138 and 203 lead to an insertion in VP-1 and VP-2, whereas insertions after the codon coding for amino acid 203 lead to insertions in VP-1, VP-2 and VP-3.

Preferred insertion sites are the positions following the amino acids that correspond to the AAV2 amino acids number 139, 161, 261, 381, 447, 453, 459, 534, 570, 573, 584, 587, 588, 657 and 713, especially 261, 453, 534, 570, 573, 587, and 588, most preferably 453 and 587. The amino acid numbers are given relative to the VP-1 amino acid sequence of AAV2.

One further embodiment of the present invention are structural proteins of parvoviruses containing insertions within the previously not described insertion sites I-453 and/or I-570.

Using I-453-based libraries may result in the selection of other peptides (as with I-587-based libraries) since adjacent residues may have an influence on the exposure and functionality of the peptides inserted into the structural protein. In addition, the sites (I-587 and I-453) are located on different loops of the AAV capsid. Thus a different mechanism of cell interaction can be assumed. Furthermore, AAV particles derived from I-453 libraries can be purified with heparin affinity chromatography, as the heparin binding site overlapping with I-587 is still intact. The same applies to other insertion sites not overlapping with I-587, preferably insertion sites I-261, I-534, I-570 and I-573.

In one potential embodiment insertions that have been selected in separate screening rounds can be combined with other insertions selected independently. For example one can use a library with an insertion of random peptides at the I-587 site for the screening method and, independently, use a second library with an insertion of random peptides at another site. Selected structural proteins of the two screening methods can then be combined by standard cloning techniques to make one clone that contains the screened insert at the respective two insertion sites.

In a further embodiment, preferred libraries contain multiple insertions at multiple sites of the structural proteins. Especially preferred libraries/structural proteins have insertions in I-453 and I-587.

By designing the sequence of the nucleic acid insert the multiplicity of the library can be controlled. The generation of such a library is for example described in WO 03/054197, hereby incorporated by reference.

The nucleic acid insert has a number of characteristics. It does not, by insertion into the coding region of the parvoviral gene, create a frame shift and thereby a truncated parvoviral structural protein. Therefore, by insertion a multimer of 3 nucleotides is inserted into the coding region of the parvoviral structural gene. The sequence is a randomly or partially randomly generated sequence, thereby generating the multiplicity of the library. A partially random sequence can for example be used to reduce the number of potential stop codons generated by insertion of the sequence and thereby reducing the number of non-functional structural mutant proteins and/or to achieve a more homogeneous distribution of the twenty different amino acids, e.g. by choosing a NNK design (with each N being any nucleotide and K standing for G or T) which in parallel reduces the number of stop codons from three to one.

In a preferred embodiment, the nucleic acid insert may contain, in addition to the randomly or partially randomly generated sequences, a further stretch of at least one codon upstream and/or downstream of the randomized or partially randomized nucleic acid sequences, preferably of 2 to 12 codons coding for small amino acids, preferably Ala, Gly, Ser, Pro, and Cys, especially an insertion of three codons for Ala upstream and two codons for Ala downstream of the randomized or partially randomized nucleic acid sequences, or an insertion of 2-5 glycin residues both, up- and downstream of the randomized or partially randomized nucleic acid sequences. Such additional amino acids do not enlarge the multiplicity of the insertion but may act as spacers to contribute to the proper accessibility of the inserted amino acids at the surface of the virions.

In a further preferred embodiment the insertion comprises linker sequences which enable a circularization of the inserted peptide sequences in order to better present the insertion. Accordingly spacer sequences are selected to form Zinc-fingers (Zn-finger), well known in the art. Preferred Zn-finger motifs are C₂H₂, C₄, and C₂HC including but not limited to motifs CX₂CX_(n)C₂, CX₂CX₁₀₋₃₀CX₂C, CX₅HX₁₀₋₃₀CX₂C, CX₂CX₁₀₋₃₀CX₄H (Laity et al., 2001 and Gamsjaeger et al., 2006).

An example of a preferred Zn-finger linker is:

X₍₃₋₅₎CXXCX₍₀₋₅₎(NNK)_(n)X₍₀₋₅₎CXXCX₍₃₋₅₎

(X=Gly or Ala, C=Cys; with each N being any nucleotide and K standing for G or T). Thus the random NNK sequence protrudes from the capsid surface.

As B-cell epitopes are composed of at least 4 amino acids (US 2004/0228798A1), in a preferred embodiment the parvovirus mutant structural protein comprises at least one insertion of 4 to 30 amino acids, preferably 5 to 20 amino acids, especially 5 to 15 amino acids. The B-cell epi-, para- or mimotopes might be composed of the inserted sequence alone, or of amino acid residues of both, the inserted peptide sequence and the viral core protein.

In a further preferred embodiment the insertion comprises within the fixed stretches upstream and downstream of the randomly or partially randomly generated sequences at least one cysteine on each side capable of forming a disulfide bond. Such a disulfide bond would spontaneously form and thereby would stabilize a loop consisting of the inserted amino acids between the two cysteines. Such loop facilitates the optimal exposure of the inserted sequence to the antibodies.

It is also an embodiment of the present invention that the parvovirus mutated structural protein comprises at least one further mutation at a different position. Such further mutation can be used to compose more complex mimotopes, to modify certain properties of the virion, e.g. it can be use to modify its natural antigenicity (e.g. (Huttner et al., 2003); WO 01/05990), to modify its chromatographic properties (e.g. WO 01/05991), to insert a second B-cell epitope, preferably a tolerogen-derived epitope, or to insert a CTL epitope. Such further mutation is selected from a point mutation, an internal or terminal deletion, an insertion and a substitution. Preferably, the further (second) insertion is internally or a N- or C-terminal fusion, whereas the further insertion has a length of 4 to 40, preferably of 5 to 30, most preferably of 7 to 20 amino acids. In one specific embodiment the insertion is a tag useful for binding to a ligand. Such tags are well known in the art, examples for such are listed in Table 3.

TABLE 3 Tags and corresponding ligands Tag Ligand HIS Nickel GST Glutathione Protein A IgG Biotin or Strep Streptavidin Calmodulin-binding peptide Calmodulin Fc-Peptide of IgG Protein A Flag GLAG- or 3xFLAG peptide HA (hemagglutinin) HA peptide

In a further preferred embodiment affinity of a identified parvovirus mutated structural protein for the binder can be modified, preferably enhanced, by generating a library of nucleic acids encoding such parvovirus mutated structural protein having a small number of random mutations per nucleic acid, at other sites than the insertion and or within the insertion, and starting the method of identifying a parvovirus mediated structural protein over again. Such process may be repeated several times, preferably 1 to 5 times, especially 1 to 2 times. A small number of random mutations in this context means an average of at least 10 sequenced clones with 1 to 10, preferably 3 to 8, especially 4 to 6 mutations compared to the starting sequence of the identified parvovirus mutated structural protein. Such random mutations can be inserted by standard techniques known in the art such as error prone PCR and DNA shuffling. In order to achieve that, the viral genomes of the mutants will be isolated and cloned into a suitable plasmid backbone. Random mutations are then inserted by e.g. error prone PCR and/or DNA shuffling. After this, a new packaging is done, followed by a genotype/phenotype coupling step and new selection for binding to a binder of choice, e.g. antibody binding.

Another embodiment of the invention is a parvovirus mutated structural protein obtainable by the methods disclosed above.

A further subject of the present invention relates to a parvovirus mutated structural protein which comprises at least one B-cell epitope heterologous to the parvovirus and not identical to a pathogen, particularly to a B-cell epitope of a pathogen, and wherein the B-cell epitope is located on the surface of the virus.

A preferred embodiment of the invention is a parvovirus mutated structural protein of the invention may be defined as described above in the context of the method of the invention. As used herein the term B-cell epitope is meant to include also mimotopes. Therefore, the epitopes can be both linear or structural. However, especially linear epitopes that are no mimotopes are preferred.

Typically, the size of a B-cell epitope is at least 4 amino acids (US 2004/0228798A1). Therefore, it is a preferred embodiment that the parvovirus mutated structural protein has an insertion consisting of at least one single or multimeric B-cell epitope of 4 to 30 amino acids, preferably 5 to 20 amino acids, especially 5 to 15 amino acids, and a further stretch of at least one amino acid upstream and/or downstream of the B-cell epitope, preferably of 2-12 amino acids selected from the group consisting of Ala, Gly, Ser, Pro, and Cys, especially 3 Ala upstream and 2 downstream of the B-cell epitope, 5 Ala upstream and 5 downstream of the B-cell epitope, or 5 Gly upstream and 5 Gly downstream of the B-cell epitope. It is preferred that such B-cell epitope is not identical to a pathogen, particularly to a B-cell epitope of a pathogen, that—in its natural environment—is accessible to a humoral immune response. Pathogen, according to this invention, means a virus, bacterium and/or eukaryotic parasite.

Such excluded B-cell epitopes of a pathogen can be identified by searching protein databases known to the skilled artisan. If the searched sequence is identical to a sequence present in a protein of a pathogen, such B-cell epitope is, according to this preferred embodiment of the invention, excluded from the invention.

In a further embodiment, the B-cell epitope heterologous to parvovirus is not identical to a mammalian (including human) or pathogen B-cell epitope, but is a functional derivative of a mammalian or pathogen B-cell epitope. A functional derivative is defined as a B-cell epitope that is identifiable e.g. by the methods according to this invention or that crossreacts with a specific monoclonal antibody for such mammalian or pathogen B-cell epitope.

In further embodiments parvovirus mutated structural proteins of the invention are further characterized as defined above, particularly wherein the tolerogen is as defined above.

In an especially preferred embodiment the parvovirus mutated structural protein comprises a B-cell epitope that is a tolerogen-derived epitope.

Preferably the B-cell epitope is a part of an antigen as defined above. Preferred antigens are IgE, tumor-antigens (e.g. Melan A, high molecular weight melanoma associated antigen (HMW MAA), CA125, IL13R, Her2/NEU, L1 cell adhesion molecule), viral receptors (CCR5), VEGF, EGFR, CD20, IL-6, IL-9, IL-13, IL-17, CETP, TNF-family members (e.g. TNF-α), or β-amyloid.

In a preferred embodiment the B-cell epitope is not a sequence previously inserted into AAV2 at position I-587/I-587 selected from the group consisting of

SEQ ID NO: 45: QAGTFALRGDNPQG, SEQ ID NO: 46: SIGYPLP, SEQ ID NO: 47: NGR, SEQ ID NO: 48: CDCRGDCFC, SEQ ID NO: 49: RGDAVGV, SEQ ID NO: 50: RGDTPTS, SEQ ID NO: 51: GENQARS, SEQ ID NO: 52: RSNAVVP, SEQ ID NO: 53: NSSRDLG, SEQ ID NO: 54: NDVRAVS, SEQ ID NO: 55: EYHHYNK, SEQ ID NO: 56: MTPFPTSNEANLGGGS, SEQ ID NO: 57: QPEHSST, SEQ ID NO: 58: VNTANST, SEQ ID NO: 59: NDVRSAN, SEQ ID NO: 60: NDVRAVS, SEQ ID NO: 61: VTAGRAP, SEQ ID NO: 62: APVTRPA, SEQ ID NO: 63: DLSNLTR and SEQ ID NO: 64: GQHPRPG, as listed in Table 4.

TABLE 4 Insertions at 587/588 of AAV2, which showed enhanced transduction on target cells (inserted in I-587 or I-588). sequence around 587/588 of wt AAV2 QRGN---------------------RQAA enhanced SEQ ID NO: 65 target transduction Ref QRGN-QAGTFALRGDNPQG------RQAA β₁ and β₃ integrin B16F10, RN22 (Girod et al., SEQ ID NO: 45 1999) QRGN- A SIGYPLP A -----------RQAA Peptide selected by HUVEC, (Nicklin et al., SEQ ID NO: 66 phage display on HSVEC 2001) HUVEC QRGN-NGR-----------------RQAA CD13 RD, KS1767 (Grifman et al., SEQ ID NO: 47 2001) QRGN- A TG CDCRGDCFC---------QAA α_(v)β₃ and α_(v)β₅ HeLa, K562, (Shi and SEQ ID NO: 67 Raji, SKOV-3, Bartlett, 2003) local appl. in vivo QRGN- AAA RGDAVGV AA --------RQAA not known MO7e (Perabo et al., SEQ ID NO: 68 selected by AAV 2003) display QRGN- AAA RGDTPTS AA --------RQAA not known MO7e (Perabo et al., SEQ ID NO: 69 selected by AAV 2003) display QRGN- AAA GENQARS AA --------RQAA not known Mec1, (Perabo et al., SEQ ID NO: 70 selected by AAV prim. B-CLL 2003) display QRGN- AAA RSNAVVP AA --------RQAA not known Mec1 (Perabo et al., SEQ ID NO: 71 selected by AAV 2003) display QRG Q R- G NSSRDLG A -----------QAA not known prim. human (Muller et al., SE Q ID NO: 72 selected by AAV coronary 2003) display endothelial cells; heart after systemic appl. QRG Q R- G NDVRAVS A -----------QAA not known prim. human (Muller et al., SEQ ID NO: 73 selected by AAV coronary 2003) display endothelial cells QRGN- AS EYHHYNK A ----------RQAA not known, selected prim. human (Work et al., SEQ ID NO: 74 by phage display saphenous 2004) on primary human vein and saphenous vein arterial SMC SMC QRGN- AS MTPFPTSNEANLGGGS A -RQAA not known, selected HUVEC, (White et al., SEQ ID NO: 75 by phage display venous endo- 2004) on HUVEC thelial cells after systemic appl. QRGN- AS QPEHSST A ----------RQAA not known, selected brain endo- (Work et al., SEQ ID NO: 76 by in vivo phage thelium after 2006) display systemic appl. QRGN- AS VNTANST A ----------RQAA not known, selected lung endo- (Work et al., SEQ ID NO: 77 by in vivo phage thelium after 2006) display systemic appl. QRG Q R- G NDVRSAN A -----------QAA not known HSaVEC (Waterkamp et SEQ ID NO: 78 selected by AAV al., 2006) display QRG Q R- G NDVRAVS A -----------QAA not known HSaVEC (Waterkamp et SEQ ID NO: 79 selected by AAV al., 2006) display QRG Q R- G VTAGRAP A -----------QAA not known Calu6 (Waterkamp et SEQ ID NO: 80 selected by AAV al., 2006) display QRG Q R- G APVTRPA A -----------QAA not known Calu6 (Waterkamp et SEQ ID NO: 81 selected by AAV al., 2006) display QRG Q R- G DLSNLTR A -----------QAA not known PC3 (Waterkamp et SEQ ID NO: 82 selected by AAV al., 2006) display QRG Q R- G GQHPRPG A -----------QAA not known H9C2 (Waterkamp et SEQ ID NO: 83 selected by AAV al., 2006) display bold: amino acid sequence of peptide insertion used to target the new receptor; italic and underlined: amino acid used as linker sequence to flank the targeting peptide; bold and double underlined: amino acid has been substituted in comparison to wild-type sequence; B16F10 = mouse melanoma cell line, RN22 = rat schwannoma, HUVEC = human umbilical vein endothelial cells, HSVEC = human saphenous vein endothelial cells, RD = rhabdomyosarcoma, KS1767 = Kaposi sarcoma, HeLa = human cercix carcinoma, K562 = human chronic myeloid leukemia in blast crisis, Raji = Burkitt lymphoma cell line, SKOV-3 = ovarian cancer, MO7e = megakaryocytic cell line, Mec1 = derived from B-cell chronic lymphocytic leukemia in prolymphoid transformation, HSaVEC = primary human venous endothelial cells, Calu6 = lung carcinoma cell line, PC3 = prostate carcinoma cell line, H9C2 = rat cardiomyoblasts

In a preferred embodiment the B-cell epitope is not a selected from the group consisting of an integrin, especially a β₁, β₃, a_(v)β₃ or a_(v)β₅ integrin, and CD13.

In a preferred embodiment the epitope is not fused to the N-terminus of the structural protein of the virus, especially not fused to the N-terminus of VP1 or VP2, whereas fusion to the N-terminus of VP3 is envisaged within this invention.

In a preferred embodiment the parvovirus mutated structural protein is capable of inducing an immunoglobulin capable of binding to the antigen the B-cell epitope is derived from.

It is an important feature, that the B-cell epitope is located on the surface of the virus.

In a preferred embodiment of the instant invention the structural protein of a parvovirus as defined above comprises an anti-idiotypic epi-/mimotope of an anti-IgE antibody, and/or an IgE epi-/mimotope.

Vaccines for the Treatment of Asthma and Allergic Diseases

Atopic asthma and allergic rhinitis are caused by adverse immune responses, typified by IgE, against otherwise harmless environmental proteins, allergens. In sensitized individuals, allergen-specific IgE becomes localized in tissues by binding to the high-affinity receptor for IgE, FcεRI, expressed by mast cells in various tissues and basophils as well as eosinophils in the blood. Subsequent encounters with the allergen result in cross-linking of IgE/FcεRI, which triggers effector cell degranulation and the release of both preformed mediators (histamine, proteolytic enzymes, and proteoglycans) and de novo synthesized mediators (prostaglandin D₂, leukotrienes, and cytokines). Together, these mediators are responsible for the clinical manifestations of allergic reactions, including hay fever, asthma, and eczema, as well as life-threatening anaphylactic reactions. Standard therapy includes inhaled corticostreroids (ICS), Beclomethasone Dipropionate (BDP), long-acting β-agonists (LABA) and leukotriene receptor antagonists (LTRAs).

The receptor-binding region of human IgE was previously mapped to the N-terminal region of the CH3 domain (Helm et al., 1988, Helm et al., 1989). Site-directed mutagenesis studies to identify the amino acid residues directly involved in the interaction have been conducted on both IgE (Presta et al., 1994) and FcεRI (Cook et al., 1997). In addition, the crystal structure of the human IgE-FcεRIα complex was recently solved by Garman and colleagues (Garman et al., 2000). The amino acid regions that are involved in receptor binding are localized in three loops and spread over most of the Cε3 domain (Pro-364, Arg-365, Arg-408, Ser-411, Lys-415, Glu-452, Arg-465, and Met-469). Binding is mediated primarily by electrostatic interaction.

Anti-IgE therapy is based on antibodies which bind the receptor-binding target domain Cε3 region of IgE, thereby preventing the binding of IgE to the FcεRI receptor and, therefore, preventing sensitization of mast cells and basophils. However, even if 99% of free IgE were neutralized by the anti-IgE antibody, the therapy still would fail because the few remaining IgE molecules would be sufficient to sensitize the respective cells. Therapeutic efficacy is provided through additional actions: FcεRI expression is regulated by the level of free IgE, in a way that reduced levels of free IgE lead to lowered densities of FcεRI on basophils and mast cells and lowered sensitivities. And, anti-IgE may lead to down-regulation of IgE production by eliminating or down-regulating IgE-expressing B cells, perhaps by cross-linking membrane-bound IgE and causing apoptosis, anergy or most likely also by complement-mediated and cell-mediated cytolysis (Wang et al., 2003). The latter mechanism was, however, not found in clinical trials performed with Omalizumab. For this monoclonal antibody, reduction of IgE production from B-cells (plasma cells) mediated by lowered IgE levels was only observed in animal and in-vitro experiments.

Most of the therapeutic monoclonal antibodies in development can only bind and neutralize free IgE or IgE associated with B-cells. In contrast, FcεRI-bound IgE is not accessible for these anti-IgE antibodies. Anti-IgE antibodies directed against regions of the IgE molecule outside of the receptor binding region (such as the variable, antigen-binding domain of IgE referred to as the IgE idiotype), can bind to an IgE molecule while it is bound to its receptor. This results in cross-linking of receptor-bound IgE, causing an anaphylactic shock in animals treated systemically with such antibodies. Importantly, except for defense mechanisms against parasite infections, IgE seems to play no role in normal physiology and IgE-deficient people are healthy with no apparent sign of pathology (Levy and Chen, 1970).

Omalizumab (XOLAIR®) is a humanized monoclonal anti-IgE antibody for passive immunization, and the first available/approved anti-IgE therapy on the market. A total of 7 phase III clinical trials were performed with this monoclonal anti-IgE antibody, which bind to the Cε3 region of IgE (for a review refer to (Bousquet et al., 2005) without crosslinking the FcεRI receptor. Omalizumab significantly reduced the rate of asthma exacerbations by 38% and the rate of total emergency visits by 47%. The efficacy of Omalizumab was unaffected by patient age, gender, baseline serum IgE or by 2- or 4-weekly dosing schedule, although benefit in absolute terms appeared to be greatest in patients with more severe asthma, defined by a lower value of percentage predicted forced expiratory volume in 1 s (FEV₁) at baseline.

As outlined before, one disadvantage of passive immunization with a monoclonal antibody is the requirement of infusions every 2-4 weeks with relatively high antibody doses making such therapies expensive. Therefore, alternative approaches are needed for the treatment of allergic diseases such as atopic allergies or asthma.

According to the present invention this problem is solved by a structural protein of a parvovirus comprising an anti-idiotypic epi-/mimotope of an anti-IgE antibody, and/or an IgE epi-/mimotope. Such structural proteins are preferably capable of forming virus-like particles. They harbor anti-idiotypic epi-/mimotopes of an anti-IgE antibody and/or IgE epi-/mimotopes on the surface of the capsid shell. Therefore, the anti-idiotypic epi-/mimotopes of an anti-IgE antibody, respectively the IgE epi-/mimotopes are accessible to the humoral immune system. Such structural protein can be used in patients to induce specifically an immune response against IgE, meaning antibodies that cross-react with IgE (anti-IgE antibodies), thereby preventing binding of IgE to its high affinity receptor FcεRI.

For a lot of the publicly available therapeutic antibodies which can be used as target antibody for AAV selection, the epitopes are not known. To be able to compare the epitopes of the target antibodies and the antibodies induced in e.g. mice after vaccination, epitope mapping can be performed. For example, epitopes recognized by anti mouse or anti human IgE antibodies can be identified from arrays using overlapping peptide scans from the respective IgE spotted on nylon membranes. Preferred antibodies are those with a binding pattern similar to that of Omalizumab, that can be used for selection of mimitopes from the AAV capsid library. Epitopes recognized by antibodies induced in e.g. mice after vaccination can be identified from arrays spotted on glass slides. Cross-reactivity of anti human IgE antibodies or antibodies induced in mice after vaccination with the constant chain regions of other Ig's can be monitored in Westernblot experiments.

Especially preferred embodiments of the invention are structural proteins of parvoviruses, especially AAV, that contain IgE epitopes or mimotopes, preferably previously known epitopes or mimotopes. As described by Rudolf, Stadler, Vogel and colleagues (Rudolf et al., 1998, Rudolf et al., 2000, Stadler et al., 1999, Vogel et al., 2000), one can develop so-called mimotope immunization vaccines based on peptide phage display libraries screened for particles recognizing BSW17, a mouse monoclonal anti-human IgE antibody. Peptide sequences best recognized by BSW17 are the mimotope sequences

(SEQ ID NO: 84) EFCINHRGYWV CGD (‘Rudolf’ (Rudolf et al., 2000)), and (SEQ ID NO: 203) INHRGYWV (‘C4M’, {Rudolf, 2000 #52}) with G, W and V (underlined) being conserved among all sequences identified (the cysteine residues (in bold) mediate a circular form of the peptide via disulfide bridging) and the epitope

VNLTWSRASG (Kricek et al., 1999). (SEQ ID NO: 85)

In the course of this invention previously novel epitopes that are especially suitable for vaccination purposes against allergic diseases like asthma have been identified:

EKQRNGTLT (‘Bind2’) (SEQ ID NO: 204) EDGQVMDVDLS (‘Flex’) (SEQ ID NO: 205) TYQCRVTHPHLPRALMR (“3DEpi1”) (SEQ ID NO: 206) RHSTTQPRKTKGSG (“3DEpi2”) (SEQ ID NO: 207) DSNPRGVSAYLSR (“3DEpi3”) (SEQ ID NO: 208) TITCLVVDLAPSK (“3DEpi4”) (SEQ ID NO: 209) KTKGSGFFVF (“C4E”) (SEQ ID NO: 210) THPHLPRALMRS (“Wang-CS”) (SEQ ID NO: 211) GETYQCRVTHPHLPRALMRSTTK (“Wang”) (SEQ ID NO: 212) LPRALMRS (C21) (SEQ ID NO: 213)

Accordingly, the present invention further relates to novel IgE B-cell epitopes Bind2, Flex, 3DEpi1, 3DEpi2, 3DEpi3, 3DEpi4, C4E, Wang-CS, Wang, and C21 and/or to a functionally active variant thereof. A functionally active variant of these epitopes means a B-cell epitope which generates in a rabbit vaccination experiment according to example 10.9 a B-cell response measurable as titer of specific antibodies binding to human IgE. The invention further relates to medicaments in general comprising such epitopes or functionally active variants thereof, preferably vaccines comprising such epitopes or functionally active variants thereof for the treatment or prevention of allergic diseases, especially asthma.

Such functionally active variants can either be single peptides or mixtures of single peptides consisting of peptide sequences of up to 40 amino acids, preferably up to 25 amino acids, more preferably 15 amino acids, especially 9 amino acids of the given sequence, or a fusion of such functionally active variant to a carrier. Such carrier is meant to be any molecule except for the naturally occurring IgE protein or part thereof (larger than the functionally active variant), preferably a parvoviral particle, but also a different virus- or bacteriophage particle, a polymer (e.g. LPH) or a fusion protein, capable of generating a B cell response (as defined above) against such functionally active variant. Such fusion to a carrier can i.e. be obtained by chemically linking the variant to the carrier or by genetically making fusion proteins or insertion variants.

These and similar sequences or parts therefore including or excluding the cysteine residues and flanking sequences can be introduced into positions I-587 and others of AAV VP as described in FIG. 14. The corresponding AAV particles can be manufactured (initially as genome-containing infectious AAV), purified and characterized. Although AAV capsids have a different conformational structure than phages, the chance that a similar structure of the mimotope sequence EFCINHRGYWVCGD (SEQ ID NO: 84) is present on both, phages and AAV, is high due to the cysteine residues building up a loop structure of the peptide sequence. For linear epitopes such as VNLTWSRASG (SEQ ID NO: 85), interchangeability should also be possible. If these AAV particles bind BSW17 (the anti-IgE antibody used for phage display), they can be used as an anti-IgE vaccine that can be used with and without co-formulation in a suitable adjuvant.

Especially preferred embodiments of the invention are structural proteins of parvoviruses that contain IgE epi-/mimotopes that, once injected into an immuno-competent mammal, induce anti-IgE specific antibodies with therapeutic efficacy without cross-linking properties. Cross-linking properties means that in an immunocompetent mammal the generated anti-IgE antibodies are binding IgE molecules in a way that IgE/FcεRI binding is still possible. By such way, and if one antibody binds several IgE molecules at a time, the high-affinity FcεRI receptor is crosslinked on effector cells leading to its degranulation. This would induce a systemic anaphylactic shock. On the other hand, the structural proteins of parvoviruses should be able to directly crosslink the respective B-cell receptor (binding the IgE epi-/mimotopes or the anti-idiotype epi-/mimotope of an anti-IgE antibody) to activate the corresponding B-cells and to induce anti-IgE antibody production independent of a T-cell response.

Vaccines for the Treatment of Alzheimer's Disease

Especially preferred embodiments of the invention are structural proteins of parvoviruses, especially AAV, that contain β-amyloid epitopes or mimotopes, preferably known epitopes or mimotopes, that can be used for the treatment of Alzheimer disease. In the context of the present invention a B-cell epitope of β-amyloid was inserted into a parvovirus capsid and displayed on the surface of the capsid. In a preferred embodiment the B-cell epitope is a human epitope. Preferably it is inserted into I-453 and/or I-587, especially into I-453 and/or I-587 of AAV1, AAV2 or AAV-6. In an especially preferred embodiment the B-cell epitope has the sequence DAEFRHDSG (SEQ ID NO: 158).

In general, misfolded proteins leading to a protein aggregation and, therefore, causing conformational diseases, are good candidate targets for an active immunization approach with AAV vaccines. Ideally, B-cell epitopes represented by misfolded proteins or protein aggregates only are chosen for presentation on AAV particles (for an overview, please refer to Uversky et al., 2006, especially; table 1-1).

Vaccines for the Treatment of Atherosclerosis

Atherosclerosis is a disease affecting arterial blood vessels. It is a chronic inflammatory response in the walls of arteries, in large part due to the accumulation of macrophage white blood cells and promoted by low density (especially small particle) lipoproteins (plasma proteins that carry cholesterol and triglycerides) without adequate removal of fats and cholesterol from the macrophages by functional high density lipoproteins (HDL). It is commonly referred to as a “hardening” or “furring” of the arteries. It is caused by the formation of multiple plaques within the arteries. There is a strong inverse relationship between the plasma concentration of cholesterol in HDLs (HDL-C) and the development of coronary heart disease (CHD). Plasma concentration of HDL-C is a powerful predictor of CHD. Although 33% of patients with CHD have low plasma levels of HDL-C as their primary lipid abnormality, there is currently no effective therapy for increasing the plasma concentration of HDL-C. Diet and moderate exercise are ineffective, statins afford only a modest 5% to 7% increase in HDL-C, and niacin has side effects and compliance profiles that limit its use.

One therapeutic approach that has been suggested for increasing plasma HDL-C concentrations is the inhibition of cholesteryl ester transfer protein (CETP) activity. CETP is a 74-kDa plasma glycoprotein that facilitates transfer of neutral lipids and phospholipids between lipoproteins and contributes to the regulation of plasma concentration of HDL-C. CETP functions in the plasma to lower the concentration of HDL-C by moving cholesteryl esters from HDLs to VLDLs and LDLs (Rittershaus et al., 2000).

Accordingly it is one embodiment of the invention to provide structural proteins of parvoviruses, especially AAV, that contain CETP epitopes or mimotopes that can be used for the treatment of atherosclerosis. Suitable epitopes or mimotopes are the human CETP derived peptides hTP10, hTP11, hTP12, hTP13, hTP18 and hTP20, hRitsch-1, hRitsch-2, hRitsch-3, hCETP-intern and hCETP C-Term:

PKTVSNLTESSSESVQS (hTP10) (SEQ ID NO: 214) SLMGDEFKAVLET (hTP11) (SEQ ID NO: 215) QHSVAYTFEED (hTP12) (SEQ ID NO: 216) INPEIITRDG (hTP13) (SEQ ID NO: 217) DISLTGDPVITASYL (hTP18) (SEQ ID NO: 218) DISLTGDPVITA (hTP20) (SEQ ID NO: 219) DQSIDFEIDSA (hRitsch-1) (SEQ ID NO: 220) KNVSEDLPLPTFSPTLLGDS (hRitsch-2) (SEQ ID NO: 221) KNVSEDLPLPT (hRitsch-3) (SEQ ID NO: 222) CDSGRVRTDAPD (hCETP-intern) (SEQ ID NO: 223) FPEHLLVDFLQSLS (hCETP C-Term) (SEQ ID NO: 224)

The present invention further relates to novel CETP B-cell epitopes hTP10, hTP11, hTP12, hTP13, hTP18, hTP20, hRitsch-1, hRitsch-2, hRitsch-3, hCETP-intern and hCETP C-Term and/or to a functionally active variant thereof. The invention further relates to medicaments in general comprising such epitopes or functionally active variants thereof, preferably vaccines comprising such epitopes or functionally active variants thereof for the treatment or prevention of atherosclerosis.

Vaccines for the Treatment of Tumor Diseases

Antibody therapies such as Herceptin, Avastin, Erbitux, Omnitarg, Rituxan, Campath, Zevalin, Mylotarg, Bexxar or Panitumumab play an increasing role in fighting various types of tumor diseases. These antibodies specifically bind epitopes of factors causing uncontrolled cellular growth, such as growth factor receptors or growth factors. Accordingly, it is a further embodiment of this invention to provide structural proteins of parvoviruses, especially AAV, that contain epitopes of such factors causing uncontrolled cellular growth.

HER2/neu (also known as ErbB-2, ERBB2) is a protein giving higher aggressiveness in breast cancers. It is a member of the ErbB protein family, more commonly known as the epidermal growth factor receptor family. HER2/neu has also been designated as CD340. HER2/neu is notable for its role in the pathogenesis of breast cancer and as a target of treatment. It is a cell membrane surface-bound receptor tyrosine kinase and is normally involved in the signal transduction pathways leading to cell growth and differentiation. Approximately 25-35 percent of breast cancers have an amplification of the HER2/neu gene or overexpression of its protein product. Overexpression also occurs in other cancer such as ovarian cancer and stomach cancer. Clinically, HER2/neu is important as the target of the monoclonal antibody trastuzumab (marketed as Herceptin).

As for an active vaccination approach, the epitope sequence QMWAPQWGPD (SEQ ID NO: 225) presented in a circular way has been shown to induce polycloncal antibodies with therapeutic effectiveness. Therefore, an Her2/NEU-AAV vaccine can be generated by insertion of the peptide

QMWAPQWGPD (SEQ ID NO: 225) into AAV using suitable adaptor sequences (Riemer et al., 2007).

Vaccines for the Treatment of Autoimmune Diseases and Chronic Inflammatory Diseases

Autoimmune diseases as well as inflammatory diseases arise from an overactive immune response of the body against substances and tissues normally present in the body. In other words, the body attacks its own cells.

Rheumatoid arthritis (RA) is an autoimmune disease which causes chronic inflammation of the joints, the tissue around the joints, as well as other organs in the body affecting 0.5-1.0% of the population in the industrialized world. It commonly leads to significant disability and consequently to a significant reduction of quality of life. If not treated appropriately, RA leads to a reduction of life expectancy (Smolen and Steiner, 2003).

Psoriasis is a chronic inflammatory disease of the skin characterized by overgrowth of epidermal cells, angiogenesis, infiltration of immune cells, and increased production of cytokines.

Similar activation of immune cells and increased production of cytokines is associated with autoimmune diseases and (chronic) inflammatory diseases as further listed below.

In order to limit or control such disease causing/related immune responses it has become an established therapeutic modality to neutralize cytokines involved in the pathogenesis of autoimmune and inflammatory diseases. Antibodies (infliximab, adalimumab) and a soluble receptor construct neutralizing the action of TNF-α (etanercept) have been established in the treatment of RA and other disease. Now there is evidence implicating several novel cytokines, including IL-32 and IL-17, in the pathogenesis of RA. In addition we assess the development of existing targets as they move towards clinical evaluation, particularly IL-1, IL-6, IL-15, IL-18 and the IL-12 superfamily (Asquith et al., 2007).

Vaccines for the Treatment of Infectious Diseases

Blocking of viral infection by induction of auto-antibodies against the cellular receptor of the virus is a suggested mechanism of a preventive or therapeutic vaccination against viruses, preferably for viruses where classical vaccination attempts have failed like HIV using CCR5 as the target receptor (Chackerian, 1999).

Accordingly, preferred embodiments of the invention are structural proteins of parvoviruses, especially AAV, that contain epitopes or mimotopes of viral receptors, preferably of CCR5, preferably known epitopes or mimotopes that can be used as vaccines for the treatment of such viral infection and associated diseases, preferably HIV infection/AIDS. In a preferred embodiment the B-cell epitope is a human epitope.

Preferred B-cell epitopes are HYAAAQWDFGNTMCQL (SEQ ID NO: 357), YAAQWDFGNTMCQ (SEQ ID NO: 358), RSQKEGLHYT (SEQ ID NO: 359) or a functionally active variant thereof.

Accordingly, preferred embodiments of the invention are structural proteins of parvoviruses, especially AAV, that contain epitopes or mimotopes of cytokines, preferably of TNF-α, IL-6 and/or IL-17, preferably known epitopes or mimotopes, that can be used for the treatment of autoimmune diseases and/or chronic inflammatory diseases, preferably rheumatoid arthritis and/or Crohn's disease. In a preferred embodiment the B-cell epitope is a human epitope. Preferably it is inserted into I-453 and/or I-587, especially into I-453 and/or I-587 of AAV1, AAV2 or AAV-6. Preferred B-cell epitopes are the human epitopes:

SSRTPSDKPVAHVVANPQAE (TNF-α V1) (SEQ ID NO: 226) SRTPSDKPVAHVVANP (TNF-α V2) (SEQ ID NO: 227) SSRTPSDKP (TNF-α V3) (SEQ ID NO: 228) NADGNVDYHMNSVP (IL-17 V1) (SEQ ID NO: 229) DGNVDYHMNSV (IL-17 V2) (SEQ ID NO: 230) RSFKEFLQSSLRALRQ (IL-6 V1) (SEQ ID NO: 231) FKEFLQSSLRA (IL-6 V2) (SEQ ID NO: 232)

The present invention further relates to novel cytokine B-cell epitopes TNF-α V1, TNF-α V2, TNF-α V3, IL-17 V1, IL-17 V2, IL-6 V1 and IL-6 V2 and/or to a functionally active variant thereof. The invention further relates to medicaments in general comprising such epitopes or functionally active variants thereof, preferably vaccines comprising such epitopes or functionally active variants thereof for the treatment or prevention of autoimmune diseases and/or chronic inflammatory diseases, preferably rheumatoid arthritis, Crohn's disease or psoriasis.

According to this invention the structural proteins of parvoviruses are the viral capsid proteins that are referred to as VP-1, VP-2 and in many instances VP-3 for most of the known parvoviruses, especially the AAV. In principal the recombinant parvoviruses made from a mutant cap gene can be used directly for vaccination in animal models or even in humans. However, as such a vaccination is a gene therapy it is preferred to use inactivated (e.g. by gamma or UV-irradiation) genome-containing AAV particles, or virus-like particles of the respective parvovirus for vaccination purposes. Such virus-like particles are capsid-like structures that are composed of the structural proteins of the respective parvovirus, e.g. VP-1, VP-2 and/or VP-3, or parts thereof such as N- or C-terminal truncated structural proteins but do not contain a viral genome. VP-2 alone has been shown to assemble into virus-like particles and can be expressed in various expression systems such as bacteria e.g. E. coli, yeasts, e.g. Saccharomyces cerevisiae, hansenula polymorpha, Pichia pastoris, in insect cells, e.g. the baculovirus expression system (SF9, SF+ or High Five cells), or in mammalian cells (such as CHO, HeLa, 293, BHK, or PerC6).

Another preferred embodiment are structural proteins of parvoviruses that do not form regular virus-like particles but capsomers or other regular or amorphous aggregates that present the foreign epi-/mimotopes in a highly structured and/or dense manner.

The parvoviral mutated structural protein can further be fused to a second protein or peptide. Such second proteins can be tags, such as provided in Table 3. Tags can for example be used for purification purposes.

Preferably the parvoviral mutated structural protein is capable of forming a multimeric structure. Accordingly, another subject of the invention relates to a multimeric structure comprising parvovirus mutated structural proteins according to the invention. Such multimeric structure can be a capsomer, a virus-like particle or a virus. Capsomers are multimeric subunits of a viral capsid, typically consisting of 5-6 capsid proteins (pentamers and hexamers). Virus-like particles are empty viruses, meaning that they do not comprise genetic material such as a viral genome or relevant part thereof.

The multimeric structure may also be an aggregate of at least 5, preferably at least 10, more preferably at least 30, most preferably at least 60 structural proteins. Compared to capsomers or virus-like particles aggregates are amorphous structures with no symmetric order.

Preferably the B-cell epitope heterologous to the parvovirus is located on the surface of the multimeric structure.

A further embodiment of the present invention is a nucleic acid coding for a parvovirus mutated structural protein of the invention such as DNA, RNA, mRNA etc.

A further embodiment of the present invention is a virus that comprises a parvovirus mutated structural protein of the invention and or nucleic acid coding for a parvovirus mutated structural protein of the invention. Such virus may be active or inactive, for example it may have been inactivated through standard techniques such as attenuation or irradiation.

A further embodiment of the present invention is a cell comprising a nucleic acid coding for the parvovirus mutated structural protein. Such cell can be a bacterium, preferably E. coli, a yeast cell, preferably S. cerevisiae, hansenula polymorpha or pichia pastoris, an insect cell, preferably SF-9, SF+ or High5, or a mammalian cell, preferably HeLa, 293, VERO, PERC6, BHK or CHO.

The parvovirus mutated structural proteins of the invention can be prepared by the method comprising the steps of (a) expressing the nucleic acid coding for the parvovirus mutated structural protein by cultivating the cell as defined above under suitable conditions, and (b) isolating the expressed parvovirus mutated structural protein of step (a).

Another subject of the invention relates to a medicament, particularly a vaccine comprising at least one parvovirus mutated structural protein of the invention and/or a nucleic acid of the invention, preferably at least one multimeric structure of the invention. Preferably, the medicament is a vaccine.

In a preferred embodiment of the invention a vaccine is a mixture of more than one multimeric structures comprising parvovirus mutated structural proteins as further defined herein. Preferably two to three virus-like particles of a parvovirus displaying different B-cell epitopes as further defined herein are combined for the vaccination of a patient. Further, it is envisaged that a vaccine according to this invention is combined with some other type of vaccine for convenience of the patient.

The medicament of the present invention may further encompass pharmaceutically acceptable carriers and/or excipients. The pharmaceutically acceptable carriers and/or excipients useful in this invention are conventional and may include buffers, stabilizers, diluents, preservatives, and solubilizers. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the (poly)peptides herein disclosed. In general, the nature of the carrier or excipients will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g. powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

In a preferred embodiment the medicament further comprises an immunostimulatory substance such as an adjuvant. The adjuvant can be selected based on the method of administration and may include mineral oil-based adjuvants such as Freund's complete and incomplete adjuvant, Montanide incomplete Seppic adjuvant such as ISA, oil in water emulsion adjuvants such as the Ribi adjuvant system, syntax adjuvant formulation containing muramyl dipeptide, or aluminum salt adjuvants. Preferably, the adjuvant is a mineral oil-based adjuvant, especially ISA206 (SEPPIC, Paris, France), most preferably ISA51 (SEPPIC, Paris, France). In another preferred embodiment the parvovirus mutated structural protein is co-formulated with at least one suitable adjuvant such as CpG, Imidazoquinolines, MPL, MDP, MALP; flagellin, LPS, LTA, or cholera toxin or derivative thereof, HSP60, HSP70, HSP90, saponins, QS21, ISCOMs, CFA, SAF, MF59, adamantanes, aluminum hydroxide, aluminum phosphate or a cytokine.

In a more preferred embodiment the immunostimulatory substance is selected from the group comprising polycationic polymers, especially polycationic peptides such as polyarginine, immunostimulatory deoxynucleotides (ODNs), peptides containing at least two LysLeuLys motifs, especially KLKLLLLLKLK, neuroactive compounds, especially human growth hormone, alumn, adjuvants or combinations thereof. Preferably, the combination is either a polycationic polymer and immunostimulatory deoxynucleotides or of a peptide containing at least two LysLeuLys motifs and immunostimulatory deoxynucleotides. In a still more preferred embodiment the polycationic polymer is a polycationic peptide.

In an even more preferred embodiment of the invention the immunostimulatory substance is at least one immunostimulatory nucleic acid. Immunostimulatory nucleic acids are e.g. neutral or artificial CpG containing nucleic acids, short stretches of nucleic acids derived from non-vertebrates or in form of short oligonucleotides (ODNs) containing non-methylated cytosine-guanine dinucleotides (CpG) in a defined base context (e.g. as described in WO 96/02555). Alternatively, also nucleic acids based on inosine and cytidine as e.g. described in WO 01/93903, or deoxynucleic acids containing deoxy-inosine and/or deoxyuridine residues (described in WO 01/93905 and WO 02/095027) may preferably be used as immunostimulatory nucleic acids in the present invention. Preferably, mixtures of different immunostimulatory nucleic acids are used in the present invention. Additionally, the aforementioned polycationic compounds may be combined with any of the immunostimulatory nucleic acids as aforementioned. Preferably, such combinations are according to the ones described in WO 01/93905, WO 02/32451, WO 01/54720, WO 01/93903, WO 02/13857 and WO 02/095027 and the AU application A 1924/2001.

In a further embodiment the medicament comprising the parvovirus mutated structural protein comprising at least one B-cell epitope heterologous to the parvovirus is (used for the manufacture of) a vaccine, preferably for preventing or treating an autoimmune disease (e.g. diabetes type 1), a tumor disease (examples are: melanoma: e.g. HMW MAA, glioblastome multiforme: e.g. CA125, anti-IL13R, colon cancer: e.g. CA125 or anti-EGF(R), breast cancer: e.g. Her2/NEU, ovarian cancer: e.g. L1 adhesion molecule, B-cell lymphoma: e.g. CD20), an allergic disease (asthma, allergies such as allergic rhinitis, examples for targets are IgE, IL-4, IL-9, IL-13), a metabolic disease (e.g. high cholesterol, intervention into the cholesterol metabolism (target example: CETP), obesity, hypertension (target example: angiotensin II), an inflammatory disease (e.g. rheumatoid arthritis, Crohn's disease; target examples: IL-6, IL-17 and TNF-α), a neurological disease (e.g. Alzheimer's disease; target example: β-Amyloid) or to be used in ophthalmology (e.g. AMD; target example: VEGF).

Also encompassed by the present inventions are methods for vaccination and/or for treating or preventing the diseases specified herein by administering to a patient an effective amount of a parvovirus mutated structural protein of the invention and or nucleic acid coding for a parvovirus mutated structural protein of the invention.

Accordingly, a further aspect of the present invention relates to a medicament of the invention for the treatment and/or prevention of

-   a) an allergic disease and/or asthma whereas the B cell epitope     comprises an anti-idiotypic epi-/mimotope of an anti-IgE antibody,     and/or an IgE epi-/mimotope, particularly a mimotope of sequence of     EFCINHRGYWVCGD or INHRGYWV, with the first G, W and V being     conserved and cysteine residues C mediating a circular form of the     peptide via disulfide bridging, or particularly an epitope selected     from the group consisting of EKQRNGTLT (SEQ ID NO: 204), EDGQVMDVDLS     (SEQ ID NO: 205), TYQCRVTHPHLPRALMR (SEQ ID NO: 206), RHSTTQPRKTKGSG     (SEQ ID NO: 207), DSNPRGVSAYLSR (SEQ ID NO: 208), TITCLVVDLAPSK (SEQ     ID NO: 209), KTKGSGFFVF (SEQ ID NO: 210), THPHLPRALMRS (SEQ ID NO:     211), GETYQCRVTHPHLPRALMRSTTK (SEQ ID NO: 212), LPRALMRS (SEQ ID     NO: 213) and a functionally active variant thereof; -   b) Alzheimer's disease whereas the B cell epitope comprises a     β-amyloid epitope or mimotope, particularly comprising or having the     sequence DAEFRHDSG (SEQ ID NO: 158) or a functionally active variant     thereof; -   c) atherosclerosis whereas the B cell epitope comprises a CETP     epitope or mimotope, particularly an epitope selected from the group     consisting of PKTVSNLTESSSESVQS (SEQ ID NO: 214), SLMGDEFKAVLET (SEQ     ID NO: 215), QHSVAYTFEED (SEQ ID NO: 216), INPEIITRDG (SEQ ID NO:     217), DISLTGDPVITASYL (SEQ ID NO: 218), DISLTGDPVITA (SEQ ID NO:     219), DQSIDFEIDSA (SEQ ID NO: 220), KNVSEDLPLPTFSPTLLGDS (SEQ ID NO:     221), KNVSEDLPLPT (SEQ ID NO: 222), CDSGRVRTDAPD (SEQ ID NO: 223),     FPEHLLVDFLQSLS (SEQ ID NO: 224) and a functionally active variant     thereof; -   d) a tumor disease whereas the B cell epitope comprises a growth     factor receptors or growth factors epitope or mimotope, particularly     a HER2/neu epitope or mimotope, especially the epitope QMWAPQWGPD     (SEQ ID NO: 225) or a functionally active variant thereof; -   e) an autoimmune disease and/or chronic an inflammatory disease,     preferably rheumatoid arthritis and/or Crohn's disease, whereas the     B cell epitop comprises an epitope or mimotope of a cytokine,     preferably of TNF-α, IL-6 and/or IL-17, especially an epitope     selected from the group consisting of SSRTPSDKPVAHVVANPQAE (SEQ ID     NO: 226), SRTPSDKPVAHVVANP (SEQ ID NO: 227), SSRTPSDKP (SEQ ID NO:     228), NADGNVDYHMNSVP (SEQ ID NO: 229), DGNVDYHMNSV (SEQ ID NO: 230),     RSFKEFLQSSLRALRQ (SEQ ID NO: 231), FKEFLQSSLRA (SEQ ID NO: 232) and     a functionally active variant thereof; or -   f) an infectious disease, preferably HIV infection, whereas the B     cell epitope comprises an epitope or mimotope of a viral receptor,     preferably of CCR5, especially an epitope selected from the group     consisting of HYAAAQWDFGNTMCQL (SEQ ID NO: 357), YAAQWDFGNTMCQ (SEQ     ID NO: 358), RSQKEGLHYT (SEQ ID NO: 359) and a functionally active     variant thereof.

In a still further aspect of the present invention the medicament of the invention as specified herein is used in a method of treating or preventing

-   a) an allergic disease and/or asthma whereas the B cell epitope     comprises an anti-idiotypic epi-/mimotope of an anti-IgE antibody,     and/or an IgE epi-/mimotope, particularly a mimotope of sequence of     EFCINHRGYWVCGD or INHRGYWV, with the first G, W and V being     conserved and cysteine residues C mediating a circular form of the     peptide via disulfide bridging, or particularly an epitope selected     from the group consisting of EKQRNGTLT (SEQ ID NO: 204), EDGQVMDVDLS     (SEQ ID NO: 205), TYQCRVTHPHLPRALMR (SEQ ID NO: 206), RHSTTQPRKTKGSG     (SEQ ID NO: 207), DSNPRGVSAYLSR (SEQ ID NO: 208), TITCLWDLAPSK (SEQ     ID NO: 209), KTKGSGFFVF (SEQ ID NO: 210), THPHLPRALMRS (SEQ ID NO:     211), GETYQCRVTHPHLPRALMRSTTK (SEQ ID NO: 212), LPRALMRS (SEQ ID     NO: 213) and a functionally active variant thereof; -   b) Alzheimer's disease whereas the B cell epitope comprises a     β-amyloid epitope or mimotope, particularly comprising or having the     sequence DAEFRHDSG (SEQ ID NO: 158) or a functionally active variant     thereof; -   c) atherosclerosis whereas the B cell epitope comprises a CETP     epitope or mimotope, particularly an epitope selected from the group     consisting of PKTVSNLTESSSESVQS (SEQ ID NO: 214), SLMGDEFKAVLET (SEQ     ID NO: 215), QHSVAYTFEED (SEQ ID NO: 216), INPEIITRDG (SEQ ID NO:     217), DISLTGDPVITASYL (SEQ ID NO: 218), DISLTGDPVITA (SEQ ID NO:     219), DQSIDFEIDSA (SEQ ID NO: 220), KNVSEDLPLPTFSPTLLGDS (SEQ ID NO:     221), KNVSEDLPLPT (SEQ ID NO: 222), CDSGRVRTDAPD (SEQ ID NO: 223),     FPEHLLVDFLQSLS (SEQ ID NO: 224) and a functionally active variant     thereof; -   d) a tumor disease whereas the B cell epitope comprises a growth     factor receptors or growth factors epitope or mimotope, particularly     a HER2/neu epitope or mimotope, especially the epitope QMWAPQWGPD     (SEQ ID NO: 225) or a functionally active variant thereof; -   e) an autoimmune disease and/or chronic an inflammatory disease,     preferably rheumatoid arthritis and/or Crohn's disease, whereas the     B cell epitop comprises an epitope or mimotope of a cytokine,     preferably of TNF-α, IL-6 and/or IL-17, especially an epitope     selected from the group consisting of SSRTPSDKPVAHVVANPQAE (SEQ ID     NO: 226), SRTPSDKPVAHWANP (SEQ ID NO: 227), SSRTPSDKP (SEQ ID NO:     228), NADGNVDYHMNSVP (SEQ ID NO: 229), DGNVDYHMNSV (SEQ ID NO: 230),     RSFKEFLQSSLRALRQ (SEQ ID NO: 231), FKEFLQSSLRA (SEQ ID NO: 232) and     a functionally active variant thereof; or -   f) an infectious disease, preferably HIV infection, whereas the B     cell epitope comprises an epitope or mimotope of a viral receptor,     preferably of CCR5, especially an epitope selected from the group     consisting of HYAAAQWDFGNTMCQL (SEQ ID NO: 357), YAAQWDFGNTMCQ (SEQ     ID NO: 358), RSQKEGLHYT (SEQ ID NO: 359) and a functionally active     variant thereof,     wherein an effective amount of the medicament is administered to a     patient in need of the prevention or treatment.

The vaccine used for immunization may be administered to a subject in need thereof, preferably mammals, and still more preferably humans, in any conventional manner, including oral, intranasal, intramuscular (i.m.), intra-lymph node, intradermal, intraperitoneal, subcutaneous (s.c.), and combinations thereof, but most preferably through intramuscular injection.

The volume of each dose for administration is preferably up to about 5 ml, still more preferably between 1 ml and 3 ml, and most preferably about 2 ml. The volume of the dose when intramuscular injection is the selected administration route is preferably up to about 5 ml, preferably up to 3 ml, preferably between 1 ml and 3 ml, more preferably between 0.5 ml and 2 ml, and most preferably about 1 ml. The amount of vaccine in each dose should be enough to confer effective immunity against and decrease the risk of developing clinical signs to a subject receiving a vaccination therewith.

Preferably, the unit dose of protein or nucleic acid should be up to about 5 μg protein/kg body weight, more preferably between about 0.2 to 3 μg, still more preferably between about 0.3 to 1.5 μg, more preferably between about 0.4 to 0.8 μg, and still more preferably about 0.6 μg. Alternative preferred unit doses could be up to about 6 μg protein or nucleic acid/kg body weight, more preferably between about 0.05 to 5 μg, still more preferably between about 0.1 to 4 μg.

The dose is preferably administered 1 to 4 times, especially 1 to 3 times, e.g. with an interval of 1 to 3 weeks. Preferred amounts of protein per dose are from approximately 1 μg to approximately 1 mg, more preferably from approximately 5 μg to approximately 500 μg, still more preferably from approximately 10 μg to approximately 250 μg and most preferably from approximately 25 μg to approximately 100 μg.

Nucleic acid delivery compositions and methods are known to those of skill in the art. The nucleic acid of the invention may be employed in the methods of this invention or in the compositions described herein as DNA sequences, either administered as naked DNA, associated with a pharmaceutically acceptable carrier or comprised in a vector. The nucleic may be administered therapeutically or as part of the vaccine composition e.g., by injection.

An “effective amount” of a nucleic acid composition may be calculated as that amount capable of exhibiting an in vivo effect, e.g. preventing or ameliorating a sign or symptoms. Such amounts may be determined by one of skill in the art. Preferably, such a composition is administered parenterally, preferably intramuscularly or subcutaneously. However, it may also be formulated to be administered by any other suitable route, including intra-nasally, orally or topically. The selection of the route of delivery and dosage of such therapeutic compositions is within the skill of the art.

Treatment in the context of the present invention refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

Examples for autoimmune disease that are especially suitable for this invention are listed in Table 5.

TABLE 5 Autoimmune diseases and suitable antibody targets/antigens Disease antibody target/antigen Myasthenia gravis Acetylcholine receptors Graves's disease Thyroid-stimulating hormone receptor Thyroiditis Thyroid Insulin-resistant diabetes Insulin receptor Asthma Beta-2 adrenergic receptors Juvenile insulin-dependent Pancreatic islet cells diabetes Pernicious anemia Gastric parietal cells Addison's disease Adrenal cells Idiopathic hypoparathyroidism Parathyroid cells Spontaneous infertility Sperm Premature ovarian failure Interstitial cells, corpus luteum cells Pemphigus Intercellular substance of skin Primary biliary cirrhosis Mitochondria Autoimmune hemolytic anemia Erythrocytes Idiopathic thrombocytopenic Platelets purpura Idiopathic neutropenia Neutrophils Vitiligo Melanocytes Osteosclerosis and Meniere's Type-II collagen disease Chronic active hepatitis Nuclei of hepatocytes Goodpasture's syndrome Basement membranes Rheumatoid arthritis Gamma globulin, virus-related antigens, IL-6, IL-17, TNF-α Sjogren's syndrome Nuclei and centromeres Systemic lupus erythematosus Nuclei, DNA, RNA, erythrocytes, etc. Scleroderma Nuclei and centromeres Polymyositis Nuclei, RNA

Preferred autoimmune diseases are asthma, Juvenile insulin-dependent diabetes (diabetes type 1) and rheumatoid arthritis. Therefore, preferred antigens are the corresponding antigens of Beta-2 adrenergic receptors, Pancreatic islet cells, Gamma globulin E, virus-related antigens IL-6, IL-17, and TNF-α.

Examples for tumor diseases that are especially suitable for this invention are listed in Table 6.

TABLE 6 Tumor diseases and suitable antibody targets/antigens Disease antibody target/antigen Melanoma HMW MAA (= high molecular weight melanoma associated antigen), BAGE, GAGE, MAGE-3, Melan A, MART-1, NY ESO, gp 100, tyrosinase Colon cancer CA125, EGFR Gliobastome multiforme (GBM) CA125, IL13R Breast cancer Her2/NEU Ovarian cancer L1 cell adhesion molecule various cancers (e.g. for colon VEGF cancer, small lung cell carcinoma) B-cell lymphoma, e.g. Non-Hodgkin CD20 Lymphoma

Examples for allergic diseases are asthma, especially atopic asthma, and all types of allergies. The preferred target antigens for vaccination against allergic diseases are IgE, IL9, and IL13, especially IgE.

An example for a metabolic disease is a disorder in the cholesterol metabolism (e.g. atherosclerosis), a preferred target antigen is CETP.

Examples for inflammatory diseases that are especially suitable for this invention are listed in Table 7.

TABLE 7 (Chronic) Inflammatory diseases Disease COPD (chronic obstructive pulmonary disease) OA (osteoarthritis) Rheumatoid arthritis Polymyalgia rheumatica Gouty arthritis, Gout, Pseudogout Atherosclerosis Crohn's disease (inflammatory bowel disease) Shoulder tendinitis, Bursitis Colitis Multiple Sclerosis Systemic Lupus Erythematosus Psoriasis Juvenile diabetes Type I diabetes mellitus (insulin-resistant diabetes) Hypothyroidism Chronic fatigue syndrome Kawasaki's disease Cardiavascular disease Pericarditis Lymph adenopathy Raynaud's phenomenon Sarcoidosis Sjogren's syndrome Spondyloarthropathies Vasculitides Scleroderma Goodpasture's syndrome Wegener's granulomatosis temporal = Giant cell arteritis Celiac disease Addison's disease Autoimmune hepatitis Grave's disease Graft-vs-host disease

Preferred target antigens are IL-6, IL-17, TNF-α and CD20.

Examples for diseases in ophthalmology are age-related macular degeneration (AMD) and diabetic retinopathy, a preferred target in these indications is VEGF.

Other preferred diseases are Alzheimer disease with the target antigen β-amyloid.

The parvovirus mutated structural protein comprising at least one B-cell epitope heterologous to the parvovirus can be especially useful for manufacture of a medicament for breaking immune tolerance.

In the context of the uses of the invention, the features of the parvovirus mutated structural protein are as defined above.

In a preferred embodiment the disease is not an infectious disease, meaning a disease caused by a virus, a bacterium, a fungus or a eukaryotic parasite.

In a further embodiment parvovirus mutated structural protein is not used to make a vector that is used in gene therapy.

In this document, the content of all cited documents is included by reference.

The following examples and figures are intended to explain the invention in detail without restricting it.

FIGURES

FIG. 1: Amino acid sequence alignment of parvoviruses AAV1, AAV-6, AAV2, AAV-3b, AAV-7, AAV-8, AAV10, AAV-4, AAV11, b-AAV, AAV-5, GPV, B19, MVM, FPV and CPV

Alignment was made using Multalin version 5.4.1 (Corpet, 1988). Symbol comparison table: blosum62, Gap weight: 12, Gap length weight: 2, Consensus levels: high=90% low=50%. Consensus symbols: ! is anyone of IV; $ is anyone of LM; % is anyone of FY; # is anyone of NDQEBZ

The corresponding amino acids to G₄₅₃ and N₅₈₇ of AAV2 and the preferred insertion range for I-453 and I-587 are boxed.

Name Length Check Weight Seq. GP-No. AAV1 799 4900 0.26 9632548 AAV-6 799 5176 0.26 2766607 AAV2 799 2359 0.50 2906023 AAV-3b 799 3639 0.50 2766610 AAV-7 799 132 0.50 22652859 AAV-8 799 3007 0.37 22652862 AAV10 799 4671 0.37 48728343 AAV-4 799 7292 0.74 2337940 AAV11 799 2546 0.74 48728346 b-AAV 799 5299 0.79 48696559 AAV-5 799 5950 1.34 91134730 GPV 799 3208 1.92 9628653 B19 799 1920 2.45 4092542 MVM 799 332 2.05 2982110 FPV 799 7156 1.61 494031 CPV 799 7674 1.61 494746 consensus 799 6436 0.00

Further parvoviruses can be found at http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fs_parvo.htm#SubFamily1.

FIG. 2: Schematic organization of the cap gene of AAV2

FIG. 3: Infection and Uptake experiment with Binder and Nonbinder Pools

For uptake wells were coated with A20 antibody and incubated with rAAV/GFP, the Binder or the Nonbinder pool (GPC of 1×10³). After removing of unbound virus, HeLa cells were seeded into the wells. After 48 h of cultivation GFP expression of the cells was analyzed by flow-cytometry. For infection HeLa cells were seeded into wells and infected with rAAV/GFP, the Binder or the Nonbinder pool (GPC of 1×10³). After 48 h of cultivation GFP expression of the cells was analyzed by flow-cytometry.

FIG. 4: Interaction of AAV variants with anti-KLH antibody

(A) 5.0×10¹⁰ and 1.0×10¹⁰ capsids of the AAV variants (H3, B6, F10, A6, D9) isolated by the screening of the AAV library with the anti-KLH mAb were dotted onto a nitrocellulose membrane. As negative control wtAAV was spotted in serial dilution ranging from 1.0×10¹⁰ to 1.6×10⁸ capsids per dot (lower lane). Likewise serial dilutions of BSA (1.0 μg-0.03 μg) were spotted on the membrane as a negative control. As a positive control different dilutions of KLH protein were spotted (1.0 μg-0.02 μg) (upper lane). The membrane was incubated with the anti-KLH antibody used for the screening of the AAV library and binding of the anti-KLH antibody to the spotted AAV variants was detected with an anti-mouse IgG (γ) HRP conjugate.

(B) After stripping of the membrane, binding of equal amounts of the AAV variants to the membrane was demonstrated using the A20 mAb and binding of the A20 mAb to the spotted AAVLPs was detected with an anti-mouse IgG (γ) HRP conjugate.

FIG. 5: Interaction of the anti-KLH antibody with a structural motif of the AAV variants

1×10¹⁰ native or heat-inactivated (10 min, 95° C.) capsids were spotted onto a nitrocellulose membrane. As negative control wtAAV was spotted ranging from 5.0×10¹⁰ to 1.6×10⁹ capsids per dot (lower lane). As a positive control different dilutions of KLH protein were spotted (1.0 μg-0.03 μg) (upper lane). The membrane was incubated with the anti-KLH antibody used for the screening of the AAV library and binding of the anti-KLH antibody to the spotted AAV variants was detected with an anti-mouse IgG HRP conjugate.

FIG. 6: Interaction of AAV variants with anti-KLH antibody (ELISA)

5×10¹⁰ AAV particles (H3, F10, B6, A6, D9) were coated onto a Maxisorp microtiter plate. As negative control wtAAV was coated ranging from 5.0×10¹⁰ to 7.8×10⁸ capsids per well (not shown). The coated particles were incubated with the anti-KLH antibody used for the screening of the AAV library. Binding of the anti-KLH antibody to the immobilized AAV variants was detected with an anti-mouse IgG HRP (horse radish peroxidase) conjugate using TMB (tetramethylbenzidine) as substrate and the absorbance was read at 450 nm. The threshold of the assay is shown as a dotted line.

FIG. 7: Interaction of AAV variants with anti-IgE antibody

5.0×10¹⁰ (left dot) and 1.0×10¹⁰ (right dot) of the AAV variants (H5, D5, E8, A9, C7, G8) isolated by the screening of the AAV library with the anti-IgE antibody (Xolair®) were spotted onto a nitrocellulose membrane (shown in boxes). Only 1.0×10¹⁰ capsids of the variant C7 were dotted. As negative control wtAAV was spotted ranging from 5.0×10¹⁰ to 3.9×10⁸ capsids per dot (lower lane). Likewise, serial dilutions of BSA (1.0 μg-0.03 μg) were spotted on the membrane as a negative control. As a positive control different dilutions of human IgE protein were spotted (1.0 μg-0.02 μg) (upper lane).

(A) The membrane was incubated with the anti-IgE antibody used for the screening of the AAV library and binding of the anti-IgE antibody to the spotted AAVLPs was detected with an anti-human IgG HRP conjugate.

(B) To demonstrate that equal amounts of AAV variants were spotted on the membrane, the membrane was stripped and spotted AAV capsids were detected using A20 mAb. Binding of the A20 mAb to the spotted AAVLPs was detected with an anti-mouse IgG (γ) HRP conjugate.

FIG. 8: Interaction of AAV variants with anti-IgE antibody vs. control antibody

5×10¹⁰ and 1×10¹⁰ particles of the AAV variants H5, E8, D5, A9, G8 (H5 only 1×10¹⁰) were dotted onto a nitrocellulose membrane. As negative control wtAAV was spotted ranging from 5.0×10¹⁰ to 1.6×10⁹ capsids per dot (lower lane). As a positive control different dilutions of human IgE or KLH protein (1.0 μg-0.03 μg) were dotted (upper lanes). The membrane was incubated with

(A) the anti-IgE antibody used for the screening of the AAV library or

(B) the control antibody (anti-KLH). Binding of the antibodies to the AAV variants was detected using the respective secondary HRP-labeled antibodies.

FIG. 9: Interaction of AAV variants with anti-CETP mAb

5.0×10¹⁰ and 1.0×10¹⁰ capsids of the AAV variants B8 and C4 isolated by the screening of the AAV library with the anti-CETP antibody were spotted onto a nitrocellulose membrane. As negative control wtAAV was spotted ranging from 5.0×10¹⁰ to 3.2×10⁹ capsids per dot (lower lane). Likewise, serial dilutions of BSA (1.0 μg-0.03 μg) were spotted on the membrane as a negative control.

(A) The membrane was incubated with the anti-CETP antibody used for the screening of the AAV library and binding of the anti-CETP antibody to the spotted AAV variants was detected with an anti-mouse IgG HRP conjugate.

(B) To demonstrate that equal amounts of AAV variants were spotted on the membrane, the membrane was stripped and spotted AAV capsids were detected using A20 mAb. Binding of the A20 mAb to the spotted AAVLPs was detected with an anti-mouse IgG (γ) HRP conjugate.

FIG. 10: Interaction of an anti-CETP antibody with the respective CETP epitope inserted into the AAV2 capsid at position 587

5.0×10¹⁰ capsids of the variants AAV-CETP-587-short and AAV-CETP-587-long and 5.0×10¹⁰ and 1.0×10¹⁰ capsids of the variants AAV-CETP-453-short and AAV-CETP-453-long were spotted onto a nitrocellulose membrane. As negative control wtAAV was spotted ranging from 5.0×10¹⁰ to 6.3×10⁹ capsids per dot. The membrane was incubated with a polyclonal anti-CETP antibody directed against the CETP epitope inserted into the AAV capsid. Binding of the anti-CETP antibody to the spotted AAV variants was detected with an anti-rabbit IgG HRP conjugate.

FIG. 11: Interaction of an anti-CETP antibody with the respective CETP epitope (CETP-intern) inserted into the AAV1 capsid after S₅₆₈ or D₅₉₀.

Serial dilutions of 1:2 ranging from 5.0×10¹¹ to 3.1×10¹⁰ capsids of the variants AAV1-CETP-588, rAAV1-GFP-CETP-588 and rAAV1-GFP-CETP-590 carrying the rabbit CETP-intern epitope were spotted onto a nitrocellulose membrane. As controls wild-type AAV1 ranging from 1.25×10¹¹ to 7.8×10⁹ capsids per dot and AAV2 with CETP insertions (CETP-intern) in 453 and 587 (AAV2-CETin-2×) ranging from 5.0×10¹¹ to 3.1×10¹⁰ capsids per dot were spotted.

(A) Membrane was incubated with an anti AAV1 intact particle antibody (Progen). Binding of the anti-AAV1 antibody to the spotted AAV variants was detected with an anti-mouse IgG HRP conjugate. (B) Membrane was incubated with a polyclonal anti-CETP antibody directed against the CETP epitope (CETP-intern) inserted into the AAV capsid. Binding of the anti-CETP antibody to the spotted AAV variants was detected with an anti-rabbit IgG HRP conjugate.

The HRP was detected by chemiluminescence using the ECL system (Pierce).

FIG. 12: ELISA for determination of binding efficiency of AAV1 to serum of AAV2 vaccinated rabbits.

Capsids were coated in equal amounts in serial dilutions from 1.0×10⁹ to 1.56×10⁷ capsids per well for 1 h at 37° C. Wells were incubated with sera from rabbits vaccinated with AAV2 (1:400 in 1% milk powder in PBS containing 0.05% Tween-20) for 1 h at 37° C. OD was measured at 450 nm.

FIG. 13: ELISA for determination of binding efficiency of AAV1 to serum of AAV2 vaccinated rabbits.

Equal amounts of capsids (1×10⁹) of rAAV2-GFP, rAAV1-GFP, rAAV1-GFP-CETP-588 and rAAV1-GFP-CETP-590 were coated onto Maxisorp 96 well plates (Nunc) and incubated with serial dilutions of sera from rabbits vaccinated with AAV2 (1:100-1:6400). OD was measured at 450 nm.

FIG. 14: Interaction of an anti-IgE antibody (Bsw17) with the respective IgE epitope inserted into the AAV2 capsid at position 587

2.5×10¹⁰ capsids of the AAV variant AAV-Kricek displaying an IgE epitope inserted at position 587 at the surface were dotted onto a nitrocellulose membrane. As negative control serial dilutions of wtAAV (5.0×10¹⁰ to 1.6×10⁹) or of the AAV variant D5 (5.0×10¹⁰ to 0.5×10¹⁰) were dotted. As a positive control human IgE was dotted ranging from 1.0 μg to 0.03 μg. The membrane was incubated with the anti-IgE mAb Bsw17 and binding of Bsw17 to the spotted AAV variants was detected with an anti-mouse IgG HRP conjugate

FIG. 15: FcεRIα expression of transfected 293 cells 293 cell clones were stably transfected with the α- and γ-chain of human FcεRI. The γ-chain is expressed under the control of an EF1α or CMV promoter. Cell surface expression of FcεRIα was analyzed by flow-cytometry using a PE-labeled anti-FcεRIα mAb.

FIG. 16: IgE binding of transfected 293 cells

The 293 cell clone D11 stably expressing the α- and γ-chain of human FcεRI was used for evaluation of IgE binding and the effect of anti-IgE antibodies (XOLAIR® or Bsw17) thereon. Cells were incubated with increasing concentrations of human biotin-labeled IgE in the absence or presence of a constant concentration of anti-IgE antibodies or a control antibody (mouse IgG₁). IgE binding was detected by flow-cytometry using PE-labeled streptavidin.

FIG. 17: Histamine release of transfected RBL2H3 cells

Rat RBL2H3 cells were stably transfected with the α-chain of human FcεRI. The stably transfected cell clone E5 was used for evaluation of histamine release induced by sensitization of the cells with human IgE and subsequent cross-linking of receptor-bound human IgE using a anaphylactic anti-human IgE antibody (Le27). Cells were sensitized with increasing concentrations of human IgE and stimulated with the anaphylactic anti-IgE antibody Le27 (▴). Histamine release was measured using a commercially available histamine ELISA. In controls cells were sensitized with increasing concentrations of human IgE without subsequent stimulation with Le27 (▪).

FIG. 18: Effect of anti-IgE mAb on histamine release

Rat RBL2H3 cells stably transfected with the α-chain of human FcεRI were sensitized with human IgE in the absence or presence of increasing concentrations of anti-human IgE mAb XOLAIR®. Histamine release was induced by cross-linking of receptor-bound IgE using the anaphylactic anti-IgE mAb Le27. Histamine release was measured using a commercially available histamine ELISA.

FIG. 19: Detection of a β-amyloid epitope displayed by AAV2 at I-587 or I-453/I-587 by β-amyloid-specific antibody

Serial dilutions (2×10¹¹-2×10⁸ capsids) of purified AAV particles displaying a β-amyloid epitope at I-587, I-453 and I-587, a CETP epitope at I-587 (as negative control) and 1 μg to 1 ng of the β-amyloid peptide (aa 1-42, Biosource, as positive control) were dotted on a membrane. The β-amyloid epitope was detected using an anti-β-amyloid mAb 6E10 (Chemicon) and as secondary antibody a peroxidase-labeled anti-mouse IgG antibody (CALTAG). Signals were detected by chemiluminescence.

FIG. 20: Induction of auto-antibodies by AAV-based vaccines vs. peptide based vaccines

Rabbits (n=2) were immunized with the AAV-based CETP vaccines AAV-TP11, AAV-TP12, AAV-TP13, or AAV-TP18 s.c. in the presence of an adjuvant. AAV-based CETP vaccines were compared with the corresponding peptide vaccines containing the same epitope coupled to LPH (Limulus polyphemus hemocyanine). The titer of CETP auto-antibodies in the immune sera was measured after the 2^(nd) (gray) and 3^(rd) (black) boost immunization.

FIG. 21: Induction of epitope specific anti-peptide antibodies by AAV-based vaccines vs. peptide based vaccines

Rabbits (n=2) were immunized with the AAV-based CETP vaccines AAV-TP11, AAV-TP12, AAV-TP13, or AAV-TP18 s.c. in the presence of an adjuvant. AAV-based CETP vaccines were compared with the corresponding peptide vaccines containing the same epitope coupled to LPH. The titer of antibodies directed against the epitope (linear peptide) in the immune sera was measured after the 2^(nd) (gray) and 3^(rd) (black) boost immunization.

FIG. 22: Induction of auto-antibodies by native and heat-denatured AAV-based vaccines

Rabbits (n=4) were immunized with native (gray) or heat-denatured (black) AAV-based CETP vaccines AAV-TP11 2× or AAV-TP18 2×s.c. in the presence of an adjuvant. The titer of CETP auto-antibodies in the immune sera was measured after the 1^(st) boost immunization.

FIG. 23: Evaluation of the impact of anti-AAV2 antibodies on immunization with AAV2-based vaccines

(A) To evaluate the impact of anti-AAV2 antibodies on the immunization success of AAV2-based vaccines, rabbits (n=3) were pre-immunized by two applications of 4.5 μg wtAAV2 (s.c. or i.m.). Serum was analyzed two weeks after 2^(nd) application for the level of anti-AAV2 antibodies. A control group (n=2) was not pre-immunized with wtAAV2.

(B) Following pre-immunization with wtAAV2 rabbits were vaccinated with the AAV2-based vaccine AAV-TP18 (7.2 μg per application). The vaccine was administered s.c. or i.m. in the presence of an adjuvant. Sera were analyzed two weeks after the 1^(st) boost vaccination for the level of CETP auto-antibodies. Results were compared to vaccination (s.c.) of animals without wtAAV2 pre-immunization.

FIG. 24: Evaluation of different prime/boost regimens for AAV-based vaccines

Three different prime/boost regimens were evaluated. Group A received one prime and three boost applications of AAV2-CETin-2× (AAV2-based vaccination). Group B received one prime and one boost immunization with AAV2-CETin-2× followed by two boost immunizations with the LPH-coupled CETP-intern peptide (LPH-peptide boost). Group C received one prime and one boost immunization with AAV2-CETIn-2× followed by two boost immunizations with AAV1-CETin (switch AAV2-/AAV1-based vaccine). Immune sera were analyzed for anti-CETP-reactivity (CETP auto-antibody titer) two weeks after the 2^(nd) (gray) and 3^(rd) boost (black) immunization.

FIG. 25: Evaluation of the impact of Montanide ISA 51 on immunization with AAV2-based vaccines

Rabbits (n=2) were immunized with the CETP vaccine AAV-TP18 i.m. or s.c. in the presence of the adjuvant Montanide ISA 51. A control group was immunized with the same vaccine s.c. in the presence of an adjuvant provided by Biogenes. Immune sera were analyzed for anti-CETP-reactivity (CETP auto-antibody titer) two weeks after the 1^(st) (white), 2^(nd) (gray) and 3^(rd) boost (black) immunization.

FIG. 26: Vaccination against CETP using AAV1 backbone

Rabbits (n=2) were immunized with AAV1 particles carrying rabbit CETP-intern epitope at position I-588. The particles (11.7 μg per vaccination) were administered i.m. at each prime or boost immunization in the presence of an adjuvant provided by Biogenes. Immune sera were analyzed for anti-CETP-reactivity two weeks after the 1^(st) (gray) and 2^(nd) boost (black) immunization.

FIG. 27: Vaccination against human β-amyloid

Rabbits (n=2) were immunized with AAV2 particles carrying a human β-amyloid epitope (aa 1-9; DAEFRHDSG, SEQ ID NO: 158) at position I-587. The particles (1 μg per application) were administered s.c. at each prime or boost immunization in the presence of an adjuvant provided by Biogenes. Immune sera were analyzed for anti-β-amyloid (Aβ(1-42) reactivity two weeks after the 1^(st) (white), 2^(nd) (gray) and 3^(rd) (black) boost immunization.

FIG. 28: Vaccination against human IgE

Rabbits (n=2) were immunized with AAV2 particles carrying a human IgE epitope (“Kricek”) at position I-587. In a control group rabbits were immunized with the same IgE epitope coupled to LPH (LPH-Kricek). Immune sera were analyzed for anti-IgE reactivity two weeks after the 1^(st) (white), 2″ (gray) and 3^(rd) (black) boost immunization. n. d.: not determined.

FIG. 29: Vaccination of rabbits with IgE derived peptides

Rabbits (n=2) were immunized with a human IgE derived epitope (GETYQSRVTHPHLPRALMRSTTK, SEQ ID NO: 236) coupled to a synthetic T-helper epitope (Wang-peptide). Another group of rabbits were immunized with a shortened variant of the epitope “Wang-CS” coupled to LPH as carrier protein (LPH-Wang-CS). Immune sera were analyzed for anti-IgE reactivity two weeks after the 2^(nd) (gray) and 3^(rd) (black) boost immunization.

FIG. 30: Evaluation of the anaphylactic properties of the anti-IgE antibodies

The effect of the anti-IgE antibodies (derived from immunization of rabbits with Wang-peptide, AAV-Kricek, AAV-3DEpi3, or AAV-Flex) on IgE mediated degranulation of basophils was investigated using RBL2H3 cells overexpressing the alpha-chain of human FcεRI. Cells were sensitized by incubation with 250 ng/ml human IgE and subsequently stimulated with polyclonal anti-IgE antibodies (total IgG fraction of immunized rabbits) at a concentration of 3 mg/ml total IgG. The anaphylactic monoclonal anti-IgE antibody Le27 (15 ng/ml) was used as positive control. Rabbit total IgG derived from unrelated immunizations (i.e. vaccinations against CETP or β-amyloid) was used as negative control. Histamine release was measured using a commercially available histamine ELISA (Neogen).

FIG. 31: Evaluation of the IgE neutralizing properties of the anti-IgE antibodies

To evaluate whether the polyclonal anti-IgE antibodies induced by vaccination of rabbits are able to neutralize IgE, the effect of the anti-IgE antibodies on IgE mediated degranulation of basophils was investigated. Human IgE (250 ng/ml) was pre-incubated with 3 mg/ml polyclonal anti-IgE antibodies (total IgG fraction of rabbits immunized with Wang-peptide, AAV-Kricek, AAV-3DEpi3 or AAV-Flex). As a positive control IgE (250 ng/ml) was pre-incubated with Xolair (1 μg/ml). Rabbit total IgG derived from unrelated immunizations (i.e. vaccinations against CETP or β-amyloid) was used as negative control. Rat basophilic RBL2H3 cells overexpressing the alpha-chain of human FcεRI were sensitized by incubation with the human IgE/anti-IgE complexes. The anaphylactic monoclonal anti-IgE antibody Le27 was used for cross-linking of receptor bound IgE. IgE-mediated histamine release was measured using a commercially available histamine ELISA.

FIG. 32: Interaction of an anti-CETP antibody with the respective CETP epitope inserted into the AAV2 capsid at different insertion sites

1.0×10¹¹ and 5.0×10¹¹ capsids of different AAV variants carrying the CETP epitope “CETP-intern” at the indicated insertion sites were dotted on a membrane (upper panel). As negative control AAV particles with the CETP epitope TP10 at position I-587 were spotted (AAV-TP10). As a positive control AAV2 variants with the CETP-intern epitope integrated at position I-453 and I-587 (AAV2-CETin-2×) were spotted (lower panel). The membrane was incubated with a polyclonal anti-CETP antibody directed against the CETP-intern epitope. Binding of the anti-CETP antibody to the spotted AAV variants was detected with an anti-rabbit IgG HRP (horse radish peroxidase) conjugate.

EXAMPLES

The following examples exemplify the invention for AAV, especially for AAV2. Due to the general similarities within the structures of the adeno-associated viruses and other parvoviruses the invention can be easily transferred to other parvoviruses.

1. Generation of an AAV Library

The cloning of the AAV library and the production of AAV capsid-modified viral particles is described by Perabo et al. (Perabo et al., 2003). The AAV library consists of approximately 4×10⁶ capsid-modified viral particles carrying random insertions of 7 amino acids at position I-587 of the AAV capsid protein. The choice of a 7-mer was empirical and was dictated by the need to insert a sequence long enough to generate an acceptable amount of diversity, but without impairing the stability of the capsid. Since typical B-cell epitopes are in general composed of 5 or 6 amino acids in length (US 2004/0228798), the peptide sequences of the library are sufficient to define B-cell epitopes that are capable to induce specific B-cell responses directed against the inserted peptide sequence when the AAV capsid variant is used as vaccine.

2. Coupling of Pheno- and Genotype of the AAV Library

The AAV library contains a pool of AAV capsid mutants which differ from each other by the random insertion of seven amino acids at position I-587 in the VP3 region of all 60 capsid proteins. When producing the AAV library, a pool of plasmids coding for the mutant capsid proteins, the viral replication proteins Rep, and harboring the inverted terminal repeats (ITRs), is introduced into 293 cells by transfection (Perabo et al., 2003).

In general, transfection of high DNA concentrations of a given plasmid pool results in the introduction of several copies per cell. Therefore, each single 293 cell takes up several different AAV plasmids all replicating in the cell and expressing AAV capsid proteins with different inserted 7mer sequences. Therefore, many transfected cells will build up a mosaic capsid composed of capsid proteins with different 7mer insertions. Since these capsids encapsulate one AAV genome being randomly chosen, many of the AAV particles will contain a vector genome which is not related to any of its 60 capsid proteins of which its capsid is composed, meaning that the geno- and phenotypes of these mosaic viruses are uncoupled. As the anti-idiotype AAV library screening approach described below is in general based on the AAV phenotype (the capsid variant of the individual AAV particles) and because the sequence information for the selected AAV variant is preferably deduced from the respective AAV genome, the coupling of geno- and phenotype is highly preferred. Therefore, a coupling step may be introduced which results in a pool of viral mutants each consisting of a viral capsid displaying only one kind of peptide insertion and containing only the respective viral genome.

To achieve replication of only one AAV mutant per cell, coupling through cell transduction with low virus concentrations was established aiming to introduce one viral genome per cell. Two different methods to transduce HeLa cells with single or low numbers of AAV particles were established: A) unspecific uptake, and B) virus infection of HeLa cells with a limited number of AAV particles.

2.1. Coupling of Geno- and Phenotype by Unspecific Uptake

The coupling of the geno- and phenotype of the AAV library was performed by unspecific AAV capsid uptake and subsequent AAV amplification by infected HeLa cells.

2.1.1. Binding of AAV to Immobilized A20 Antibody

One cell culture plate (Ø15 cm, TPP) was coated with 10 ml AAV2 capsid-specific A20 antibody (supernatant of respective hybridoma) for 2 h at room temperature. The A20 antibody binds to intact AAV capsids (Grimm et al., 1999, Wistuba et al., 1997) independently from the sequence inserted in position I-587. The A20-coated plates were washed three times with 20 ml D-PBS containing 1% Tween-20 to remove unbound A20 antibody. After washing the coated plates were incubated with 20 ml blocking buffer (5% milk powder in D-PBS containing 1% Tween-20) for 2 h at room temperature to avoid unspecific binding of the AAV particles to the plates.

The plates were then incubated with the AAV library at genomic particles per cell (GPC) of 10, 100 and 1000 in a total volume of 10 ml blocking buffer for 2 h at room temperature. The genomic titer of the AAV population had been determined by quantitative real-time PCR as described below. After incubation of the A20-coated plates with the AAV library, unbound virus was removed by 20 washes with 10 ml D-PBS/1% Tween-20 followed by four washes with 10 ml D-PBS.

2.1.2. Uptake and Amplification of AAV by HeLa Cells

4.0×10⁶ HeLa cells per Ø15 cm culture plate were seeded onto the AAV particles captured by the A20 antibody. Simultaneously, HeLa cells were infected with Adenovirus Type-2 (AdV2) at a MOI of 5 to induce replication of AAV particles. Infection and cultivation of the HeLa cells was performed in a total volume of 10 ml DMEM containing 10% (v/v) fetal calf serum (FCS) and 1% (v/v) Penicillin/Strepto-mycin for 24 h at 37° C. and 5% CO₂ in a humidified atmosphere. After 24 h of cultivation another 10 ml of DMEM containing 10% (v/v) FCS and 1% Penicillin/Streptomycin was added to the plate to a total volume of 20 ml. Cells were cultured for an additional 24 h at 37° C. and 5% CO₂ in a humidified atmosphere. After 24 h of cultivation, HeLa cells were harvested using a cell scraper and collected by centrifugation (3000 g, 10 min, 4° C.). Cells were washed with 5 ml D-PBS. After centrifugation (3000 g, 10 min, 4° C.) the cell pellet was resuspended in 500 μl lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5). Cells were lyzed by three thaw/freeze cycles using liquid nitrogen and a thermoblock tempered at 37° C. The cell lysate was treated with 50 U/ml benzonase (Merck) for 30 min at 37° C. After benzonase treatment the cell lysate was cleared by centrifugation (3700 g, 4° C., 20 min).

2.1.3. Evaluation of AAV Genomic Titers by Light Cycler PCR

For determination of genomic titers 50 μl of virus containing benzonase-treated cell lysate was used for isolation of DNA. For inactivation of AdV the lysate was incubated at 60° C. for 30 min. The lysate was diluted four-fold with PBS and total DNA was purified using the DNeasy Tissue Kit including a Proteinase K treatment (Qiagen). DNA was eluted in 200 μl Tris-HCl, pH 7.5. 2.0 μl DNA were applied to the Light Cycler PCR Master Mix using the Light Cycler FastStart DNA Master SYBR Green I Kit (Roche). Primers

4066-Back (SEQ ID NO: 86) 5′-ATG TCC GTC CGT GTG TGG-3′ and 3201-For (SEQ ID NO: 87) 5′-GGT ACG ACG ACG ATT GCC-3′ were used for PCR amplification. Titers were determined by computer evaluation using the program provided with the Roche Light Cycler 2.0 and compared to a standard.

2.1.4. PCR Amplification and Subcloning of the AAV Library Insertion Site

To analyze the coupling of the geno- and phenotype of the AAV library after the unspecific up-take and amplification of AAV by the HeLa cells, the AAV library DNA containing the insertion site was amplified by PCR, subcloned into pRC-Kotin (described below) and analyzed by sequencing. Therefore, total DNA was purified from a 50 μl aliquot of the transduced HeLa cell lysate as described above. The cell lysate was diluted fourfold in PBS and total DNA was prepared using the DNeasy Tissue Kit according to the instructions of the manufacturer. Total DNA was eluted in 50 μl H₂O. The fragment of the AAV genome containing the library insertion site was amplified by PCR using 5.0 μl of the total DNA prepared from the cell lysate as template and 20 pmol of the primers

BsiWI back (SEQ ID NO: 88) 5′-TAC CAG CTC CCG TAC GTC CTC GGC-3′ and SnaBI forward (SEQ ID NO: 89) 5′-CGC CAT GCT ACT TAT CTA CG-3′ in a total volume of 50 μl. PCR was performed using the High Fidelity Platinum Pfx Polymerase Kit (Invitrogen). After an initial heat denaturation of the DNA template at 95° C. for 3 min, DNA was amplified by 35 PCR cycles (45 sec at 95° C. denaturation, 40 sec at 56° C. annealing, 2 min at 68° C. extension). Amplification was terminated after a final extension step at 68° C. for 10 min. An aliquot of the PCR reaction was analyzed on a 1% TBE agarose gel. The PCR product was purified using the PCR Purification Kit (Qiagen). The PCR product was cloned into the BsiWI/SnaBI site of the vector pRC-Kotin. The pRC plasmid was previously described (Girod et al. 1999). In pRC-Kotin the ITRs have been removed and an additional SnaBI restriction site was introduced downstream of the Cap ORF. Electro-competent E. coli XL-1 Blue MRF were transformed with the vectors by electroporation. The plasmids of 100 single independent clones of the cloning reaction were prepared and the insertion site of the library was sequenced using the primer

4066-back SEQ ID NO: 86 5′-ATG TCC GTC CGT GTG TGG-3′. 2.1.5. Statistical Analysis of the AAV Library Sequences after Unspecific Uptake by HeLa Cells

The nucleotide sequences obtained from sequencing of at least 100 plasmids of single clones were translated into protein sequences and the 7mer peptide sequence inserted at position I-587 of AAV2 cap was analyzed. The state of geno- and phenotype coupling of the AAV2 library is reflected by the amount of stop codons detected within the 7mer peptide sequence inserted at position I-587. Since sequences encoding stop codons in-frame with the capsid protein can only be assembled in intact AAV capsids if more than one capsid encoding plasmid was transfected into one HeLa cell. Regarding the codon-usage, 14.6 stop codons in one hundred 7mer peptide sequences are statistically expected (due to the NNB design of the library), and 8.6 out of a hundred occurred in the original non-coupled AAV library, whereas 9.0 stop codons were found in average in the respective AAV DNA library.

Considering the number of stop codons as an indicator for the coupling state of the library, the number of stop codons should be markedly decreased after pheno-/genotype coupling of the library. In addition, the biodiversity of the library should be maintained. An indicator for the biodiversity is the absence of duplicate sequences.

Regarding single sequences about 40% of sequences occurred more than once after AAV uptake at GPC 10, which is to be regarded as a reduced biodiversity. In the uptake experiments utilizing GPC 100 and 1000 there were no duplicate sequences pointing to a better ratio between genomic particles and cells and a better diversity. The number of stop codons was lower as in the original library, which points to a well coupled library (Table 8). The number of stop codons calculated per 100 sequences increased as expected, when higher GPC were used, since in case of GPC 1000 it was very likely that more than one viral mutant was able to be taken up by one cell. Taken together the uptake with GPC of 100 is appropriate in terms of the coupling of pheno- and genotype and the maintenance of an adequate diversity of the AAV library.

TABLE 8 Frequency of stop codons after coupling by uptake (GPC 10, 100 and 1000): At least 100 sequences were analyzed and the number of stop codons was calculated per 100 sequences. viral pool uptake uncoupled GPC 10 GPC 100 GPC 1000 stop codons. per 8.6 1.4 2.0 4.6 100 seq.

2.2. Coupling of Geno- and Phenotype by Infection

Coupling of an AAV library by infection without loss of biodiversity will work, if each mosaic virion from a non-coupled AAV library contains at least one cell binding motif which renders the AAV particle infectious. Alternatively, if e.g. only each 10th particle is still infectious (due to low abundance of corresponding binding and intracellular trafficking motifs), a 10 fold excess of particles has to be processed to ensure that each sequence from the library is taken up by a cell at least once as the likelihood is proportionally augmented that each genome is packaged at least into one infectious particle. As for the uptake experiment different GPCs were tested to determine the optimal coupling efficiency retaining full biodiversity of the AAV library.

2×10⁶ HeLa cells were seeded in 15 ml medium (DMEM containing 10% (v/v) FCS and 1% Penicillin/Streptomycin) in 015 cm cell culture plates (TPP) and cultivated for 24 h at 37° C., 5% CO₂ in a humidified atmosphere. After 24 h medium was changed and the cells were infected with AAV genomic particles per cell (GPC) of 10, 100 and 1000 and incubated for 48 h in the presence of adenovirus (MOI 5) to allow replication and packaging of AAV. HeLa cells were harvested using a cell scraper and collected by centrifugation (3000 g, 10 min, 4° C.). Cells were washed with 5 ml D-PBS. After centrifugation (3000 g, 10 min, 4° C.) the cell pellet was resuspended in 500 μl lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5). Cells were lyzed by three thaw/freeze cycles using liquid nitrogen and a thermoblock tempered at 37° C. The cell lysate was cleared by centrifugation.

Total DNA was purified, viral DNA amplified by PCR and cloned into the AAV pRC-Kotin vector as described above. Plasmids were transformed into bacteria and single clones were picked and sequenced as described above.

2.2.1. Statistical Analysis of the AAV Library Sequences after Infection of HeLa Cells

The nucleotide sequences obtained from sequencing of at least 100 plasmids of single clones were translated into protein sequences and the 7mer peptide sequence inserted at position I-587 of AAV2 VP was analyzed. As described above (2.1.5) the state of geno- and phenotype coupling of the AAV library is reflected by the amount of stop codons detected within the 7mer peptide sequence inserted at position I-587.

As observed for the coupling by unspecific uptake a comparatively high number of sequences occurred more than once when a GPC of 10 is used for infection of HeLa cells with the AAV library. The diversity of the library was higher when GPCs of 100 and 1000 were used for infection of HeLa cells with the AAV library, since no duplicate sequences were identified among at least 100 analyzed sequences. The number of stop codons, as an indicator for the state of geno- and phenotype coupling, was down to zero with GPCs of 100 and 1000 (Table 9) demonstrating the efficient coupling of pheno- and genotype of the library.

TABLE 9 Frequency of Stop codons in infection experiment with GPC 10, 100 and 1000: At least 100 sequences were analyzed and the number of stop codons was calculated per 100 sequences. viral pool infection uncoupled GPC 10 GPC 100 GPC 1000 stop codons. per 8.6 1.2 0 0 100 seq.

2.3. Coupling of Geno- and Phenotype by Limited Dilution

In addition to the coupling methods described above (uptake or infection), the coupling of the geno- and phenotype of the AAV library can be performed by transfection of HeLa cells with a limited number of library plasmids. The amount of plasmids used for transfection is either calculated so that statistically only one single plasmid is taken up by each HeLa cell and finally entering the nucleus, or, the ideal number of AAV library genomes is determined with following model read-out system:

A self-replicating (e.g. B1/EBNA or SV40ori/large-T antigen) reporter gene plasmid (such as GFP) is transfected in increasing amounts together with a non-relevant carrier DNA such as pUC19, keeping the total DNA amount constant. The use of a self-replicating plasmid system ensures that each transfected cell produces enough GFP to be detected in a flow-cytometry assay. Fluorescence per cell and percent GFP positive cells define a crossing point, where increasing copy numbers of the reporter gene plasmid are no more proportional to an increase of GFP positive cells and where the fluorescence per cell is increasing indicating the uptake of more than one single reporter gene plasmid per cell. The amount of reporter gene plasmid respective library plasmid below the concentration at the crossing point has to be chosen to ensure the uptake of at maximum one library plasmid per cell.

Therefore, after infection with adenovirus each transfected cell produces only one defined type of AAV variant corresponding to the library plasmid that was taken up by the cell.

3. Evaluation of Unspecific-Uptake of AAV by HeLa Cells

Since the random peptide sequence of the AAV library is introduced at position 587 of the AAV capsid comprising the heparin binding domain of AAV, the AAV variants can be differentiated into variants that still bind to heparin due to reconstitution of the binding motif by the inserted random peptide sequence (Binder) and variants that do not bind to heparin (Nonbinder).

An AAV helper plasmid containing random peptides inserted into cap (helper plasmid library) was co-transfected with a double-stranded GFP vector plasmid to generate a GFP vector virion library. This library was coupled by infection. This coupled library was applied to a heparin affinity column to separate heparin binding from non-binding variants. For this, the library was applied to a heparin column (HiTrap, Amersham Bioscience). The flow-through contained the Nonbinders, whereas the Binders were bound to the column and then eluted from the column by 1M NaCl. Then both fractions were purified by Iodixanol step gradient centrifugation to concentrate the virions. Thereafter, genomic titers of both pools were determined by Light Cycler PCR. After the purification step genomic titers of 1×10⁷ per μl (500 μl total) were obtained.

Infection and uptake experiments on HeLa cells with the Binder and the Nonbinder pools should reflect the different capabilities of the variants to enter the cells. Binders and Nonbinders were expected to show clear differences regarding their infectivity due to the different heparin-binding properties and the ability to interact with HSPG. In contrast, Binders and Nonbinders were expected to show no major differences regarding their transduction efficacy in uptake experiments, since uptake was assumed to be independent form HSPG and a heparin binding motif.

To analyze this, 5.0×10⁴ HeLa cells/well were seeded into a 24-well cell culture plate in a volume of 0.5 ml medium (DMEM with 10% (v/v) FCS and 1% (v/v) Penicillin/Streptomycin). After cultivation of the cells for 1 d at 37° C. in a humidified atmosphere containing 5.0% CO₂, cells were infected with 1×10⁸ genomic particles per well (GPC 1×10³) of the Binder/GFP, Nonbinder/GFP pool or rAAV/GFP (recombinant wtAAV encoding GFP as a control). After 48 h of cultivation at 37° C. in a humidified atmosphere containing 5.0% CO₂ GFP expression levels of the cells were determined by flow cytometry (FIG. 3). For the uptake experiments, 24-well plates were coated with 100 μl/well A20 antibody (hybridoma supernatant recognizing the intact AAV capsid) for 1 h at room temperature. Unbound A20 was removed by 10 washes using D-PBS/1% Tween-20. Wells were blocked by incubation with 0.5 ml/well blocking buffer (10% milk powder in D-PBS/1% Tween-20) for 2 h at room temperature. A20-coated wells were incubated with rAAV/GFP, the Binder library or the Nonbinder library with 1×10⁸ genomic particles per well (GPC 1×10³). After incubation for 1 h at room temperature, unbound AAV particles were removed by 10 washes using D-PBS/1% Tween-20 followed by 4 washes with D-PBS. Then 1.0×10⁵ HeLa cells/well were seeded on top of the bound AAV particles in a volume of 0.5 ml medium (DMEM with 10% FCS and 1% Penicillin/Streptomycin) and incubated for 48 h at 37° C. in a humidified atmosphere containing 5.0% CO₂. Transduction efficiency (GFP expression of the cells) was determined by flow cytometry (FIG. 3).

As expected, the Binder pool and rAAV/GFP showed comparable transduction efficacies in the infection experiments, whereas the infectivity of the Nonbinder pool was strongly reduced. The residual 20% transduction efficiency observed for the Nonbinder pool in the infection experiments is most probably mediated by HSPG independent pathways such as makro- or pinocytosis or alternative receptors.

In contrast to the infection experiments, the transduction efficacy of the Binder and Nonbinder pool was found to be comparable in the uptake experiments.

These data demonstrate that in contrast to infection the uptake of AAV variants by HeLa cells is independent from the heparin binding domain and independent from the peptide sequence inserted at position 587 of the AAV capsid.

4. Production and Purification of AAV Variants 4.1. AdV Helper Plasmid

An AdV helper plasmid encoding AdV E2, E4 and VAI-VAII was used for AAV manufacturing in 293 or 293T cells. The helper plasmid pUCAdvE2/E4-VAI-VAII was constructed by subcloning of the BamHI restriction fragment encoding the adenovirus E2 and E4-ORF6 from pAdEasy-1 into the site BamHI site of pUC19. The resulting plasmid is referred to as pUCAdVE2/E4. The VAI-VAII fragment from pAdvantage was amplified by PCR using the primers

XbaI-VAI-780-3′ (SEQ ID NO: 90) 5′-TCT AGA GGG CAC TCT TCC GTG GTC TGG TGG-3′ and XbaI-VAII-1200-5′ (SEQ ID NO: 91) 5′-TCT AGA GCA AAA AAG GGG CTC GTC CCT GTT TCC-3′, cloned into pTOPO and then subcloned into the XbaI site of pUCAdvE2/E4. The resulting plasmid pUCAdvE2/E4-VAI-VAII (in short pUCAdV) was evaluated in co-transfection experiments for production of AAV as described below. AAV particle formation was analyzed using the A20 ELISA.

4.2. Production of AAV Variants by Co-Transfection of HEK 293 T-Cells

For production of AAV particles HEK 293-T cells were co-transfected with the vector plasmid pRC-Kotin containing the subcloned library insertion sequence, pGFP and the helper plasmid pUCAdV (described above). The plasmid pGFP contains a GFP (green fluorescent protein) cDNA under the control of a CMV promoter. This GFP cassette is flanked with AAV derived ITRs. Therefore, co-transfection of 293-T cells with these three plasmids will result in the production of AAV particles displaying the library 7mer sequence at the surface and containing the GFP cassette with ITRs as viral genome.

AAV variants obtained by the direct cloning approach (described below) were produced as described above with the following modification. For co-transfection of the vector plasmid pUCAV2 containing the epitope/mimotope (in I-453 or I-587) and pUCAdV a molar ratio of the plasmids of 1:1 was chosen. For Calcium phosphate transfection of one culture plate with 293-T cells using the Calcium phosphate transfection protocol as described above, 12.0 μg pUCAV2 (containing the epitope/mimotope in I-453 or I-587) and 24.0 μg pUCAdV were used. Transfection was performed as described above.

For co-transfection 7.5×10⁶ 293-T cells were seeded into each 015 cm cell culture plate in a total volume of 17.5 ml medium (DMEM containing 10% FCS, 5 mM L-Gln and ABAM) 24 h before transfection and cultivated at 37° C., 5% CO₂ in a humidified atmosphere. For co-transfection of pRC-Kotin, pGFP and pUCAdV a molar ratio of the plasmids of 1:1:1 was chosen. For Calcium phosphate transfection of one culture plate with 293-T cells using the Calcium phosphate transfection protocol as disclosed in US 2004/0053410, 9.0 μg pRC-Kotin, 9.0 μg pGFP and 18.0 μg pUCAdV were mixed in 875 μl 270 mM CaCl₂. In brief, 875 μl 2×BBS (50 mM BES (pH 6.95), 280 mM NaCl and 1.5 mM Na₂HPO₄) was added to the mixture and the resulting solution was carefully mixed by pipetting. The solution was incubated for 20 min at room temperature and then added drop-wise to the cell culture plate. Cells were incubated at 35° C., 3% CO₂ in a humidified atmosphere for 18 h. After 18 h at 35° C. and 3% CO₂ cells were cultivated for an additional 3d at 37° C., 5% CO₂ in a humidified atmosphere.

293-T cells were harvested with a cell lifter, transferred into 50 ml plastic tubes (Falcon) and centrifuged at 3000 g at 4° C. for 10 min. The cell pellet was resuspended in 1.0 ml lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5) and objected to three rounds of freeze and thaw cycles. The lysate was treated with 100 U/ml benzonase (Merck) at 37° C. for 30 min. The cell lysate was cleared by two centrifugation steps (3700 g, 4° C., 20 min) and the AAV-containing supernatant was used for further purification.

The AAV capsid titer of the lysate was determined using a commercially available ELISA (AAV Titration ELISA, Progen).

4.3. Purification of AAV Particles by Density Gradient Centrifugation Using Iodixanol

AAV particles were purified by iodixanol gradient centrifugation. The virus-containing cell lysate was cleared by centrifugation (3700 g, 4° C., 20 min) and the cleared lysate was transferred to Qickseal ultracentrifugation tubes (26×77 mm, Beckman). Iodixanol solutions (Sigma) of different concentrations were layered beneath the virus containing lysate. By this an Iodixanol gradient was created composed of 6.0 ml 60% on the bottom, 5.0 ml 40%, 6.0 ml 25% and 9.0 ml 15% Iodixanol with the virus solution on top. The gradient was spun in an ultracentrifuge at 416.000 g for 1 h at 18° C. The 40% phase containing the AAV particles was then extracted with a cannula by puncturing the tube underneath the 40% phase and allowing the solution to drip into a collecting tube until the 25% phase was reached. The AAV capsid titer of the 40% phase was determined using a commercially available ELISA (AAV Titration ELISA, Progen).

5. Selection of AAV Particles with Specific Affinity for a Target Antibody from the Coupled Viral Library

5.1. Anti-Idiotype Selection Using an Anti-KLH Antibody

To proof the concept of selection of anti-idiotype AAV variants, an anti-KLH (Keyhole Limpet Hemocyanin) mouse monoclonal antibody (R&D Systems) was used as selection antibody. The mouse anti-KLH monoclonal antibody (IgG₁ isotype) was obtained from a mouse immunized with purified KLH as antigen. In another approach unspecific binding of AAV particles to the cell culture plate in the absence of an immobilized selection antibody was analyzed (negative control). In the experiments described in this example, an AAV library was used, whose geno- and phenotype was coupled by infection at GPC1000 as described above (2.2)

5.1.1. Binding of AAV to Immobilized Anti-KLH Antibody Vs. Binding of AAV to Uncoated Cell Culture Plate

A cell culture plate (Ø10 cm, TPP) was coated with 5 ml anti-KLH monoclonal IgG₁ antibody at a concentration of 10 μg/ml in coating buffer (0.8 ml 0.2M NaHCO₃, 1.7 ml 0.2M Na₂CO₃ ad 10 ml H₂O) for 18 h-24 h at 4° C. In another approach (negative control) plates were treated with coating buffer in the absence of an antibody. All plates were washed three times with 10 ml D-PBS containing 1% Tween-20. After washing the plates were incubated with 10 ml blocking buffer (5% milk powder in D-PBS containing 1% Tween-20) for 2 h at room temperature to avoid unspecific binding of the AAV particles to the plate. The plate was then incubated with 1×10⁸ genome-containing AAV library particles in a total volume of 5 ml blocking buffer for 2 h at room temperature. The genomic titer of the AAV population was determined by quantitative real-time PCR as described above. After incubation of the anti-KLH mAb-coated plate or uncoated plate (negative control) with the AAV library, unbound virus was removed by 20 washes with 10 ml D-PBS/1% Tween-20 followed by four washes with 10 ml D-PBS.

5.1.2. Uptake and Amplification of AAV by HeLa Cells

1.0×10⁶ HeLa cells per plate were seeded onto the AAV particles captured by the anti-KLH mAb or adsorbed by the plate in an unspecific way in the control approach (negative control). Simultaneously, HeLa cells were infected with Adenovirus Type-2 (AdV2) at a MOI of 5 to induce replication of AAV particles. Infection and cultivation of the HeLa cells was performed in a total volume of 10 ml DMEM containing 10% (v/v) fetal calf serum (FCS) and 1% (v/v) Penicillin/Streptomycin for 48 h at 37° C. and 5% CO₂ in a humidified atmosphere. After 48 h of cultivation, HeLa cells were harvested using a cell scraper and collected by centrifugation (3000 g, 10 min, 4° C.). Cells were washed with 5 ml D-PBS. After centrifugation (3000 g, 10 min, 4° C.) the cell pellet was resuspended in 250 μl lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5). Cells were lyzed by three freeze/thaw cycles using liquid nitrogen and a thermoblock tempered at 37° C.

5.1.3. PCR Amplification and Subcloning of the AAV Library Insertion Site

Total DNA was purified from a 50 μl aliquot of the transduced HeLa cell lysate. The cell lysate was diluted fourfold in PBS and total DNA was prepared using the DNeasy Tissue Kit according to the instructions of the manufacturer. Total DNA was eluted in 50 μl H₂O. The fragment of the AAV genome containing the library insertion site was amplified by PCR using 5 μl of the total DNA prepared from the cell lysate as template and 20 pmol of the primers

BsiWI back (SEQ ID NO: 92) 5′-TAC CAG CTC CCG TAC GTC CTC GGC-3′ and SnaBI forward (SEQ ID NO: 93) 5′-CGC CAT GCT ACT TAT CTA CG-3′

in a total volume of 50 μl. PCR was performed using the High Fidelity Platinum Pfx Polymerase Kit (Invitrogen). After initial heat denaturation of the DNA template at 95° C. for 3 min, DNA was amplified by 35 PCR cycles (45 sec at 95° C. denaturation, 40 sec at 56° C. annealing, 2 min at 68° C. extension). Amplification was terminated after a final extension step at 68° C. for 10 min. An aliquot of the PCR reaction was analyzed on a 1% TBE agarose gel. The PCR product was purified using the PCR Purification Kit (Qiagen). The PCR product was cloned into the BsiWI/SnaBI site of the vector pRC-Kotin. Electro-competent E. coli XL-1 Blue MRF were transformed with the vectors by electroporation. The plasmids of 100 single clones were prepared and the insertion site of the library was sequenced using the primer

4066 back (SEQ ID NO: 86) 5′-ATG TCC GTC CGT GTG TGG-3′.

The obtained nucleotide sequences were translated into protein sequences and the 7mer peptide sequence inserted at position I-587 of AAV2 VP was analyzed. The results are summarized in Table 10. AAV particles containing the same peptide sequence at the library insertion site as AAV particles obtained by screening of the library using the uncoated culture plates (negative control) were considered as non-specifically bound particles and were excluded from further analysis.

TABLE 10 AAV Variants identified in the Library Screening approach AAV selection frequency variant antibody sequence round I round II round III round IV H3 anti-KLH ARAGLPG 20.9 0.0 N/A N/A SEQ ID NO: 94 B6 anti-KLH LRPDARP 15.4 50.0 N/A N/A SEQ ID NO: 95 A6 anti-KLH PRTDSPR 26.4 45.0 N/A N/A SEQ ID NO: 96 F10 anti-KLH PTLTPPR 19.8 0.0 N/A N/A SEQ ID NO: 97 D9 anti-KLH STLAPPA 2.2 0.0 N/A N/A SEQ ID NO: 98 C4 anti-CETP SRPPNPA 73.2 22.2 33.3 N/A SEQ ID NO: 99 B8 anti-CETP MGSPSTR 0.0 33.3 33.3 N/A SEQ ID NO: 100 E2 anti-CETP RDHPGIR 0.0 0.0 29.8 N/A SEQ ID NO: 101 B6 anti-CETP VGSPSTR 0.0 0.0 3.5 N/A SEQ ID NO: 102 A2 anti-CETP LPTARSP 2.8 0.0 0.0 N/A SEQ ID NO: 103 C7 anti-IgE VYSPTGK 0.0 8.1 84.0 97.4 SEQ ID NO: 104 D5 anti-IgE SDAPLPR 65.2 86.0 0.0 0.0 SEQ ID NO: 105 H5 anti-IgE ETQLRAT 0.0 72.7 17.1 0.0 SEQ ID NO: 106 E8 anti-IgE GLGTQPR 0.0 0.0 22.9 61.5 SEQ ID NO: 107 G8 anti-IgE DKTGSKP 23.8 0.0 0.0 0.0 SEQ ID NO: 108 A9 anti-IgE TSASRAP 0.0 0.0 12.0 0.0 SEQ ID NO: 109 E11 anti-IgE ACAPTGV 0.0 0.0 5.7 0.0 SEQ ID NO: 110

5.1.4. Second Round of Anti-KLH mAb Screening

The number of genomic particles (genomic AAV titer) contained in the HeLa cell lysate was determined by quantitative real-time PCR (see 2.1.3). For the second round of selection, cell culture plates were coated with anti-KLH mAb or were left uncoated (negative control) as described above. Blocking and washing of the plates was performed as describe above. Plates were incubated with the volume of HeLa cell lysate (containing the AAV pool of the first selection round) corresponding to GPC of 100 in a total volume of 5 ml blocking buffer. After incubation of the plates with the AAV pool obtained from the first round of selection for 2 h at room temperature, unbound virus was removed by 20 washes with 10 ml D-PBS/1% Tween-20 followed by four washes with 10 ml D-PBS. Uptake and amplification of the anti-KLH mAb bound AAV or non-specifically bound AAV (negative control) by HeLa cells was performed as described above. Preparation of total DNA, PCR amplification and subcloning of the AAV library insertion site was performed as described above. The results are summarized in Table 10. AAV particles containing the same peptide sequence at the library insertion site as AAV particles obtained by screening of the library using the uncoated culture plates were considered as non-specifically bound particles and were excluded from further analysis.

5.1.5. Characterization of AAV Particles Obtained by Anti-KLH Screening of the AAV Library

AAV particles of the library screening approach were produced and purified as described above. AAV capsid titers were analyzed using the AAV titration ELISA.

Dot Blot Analysis

The AAV capsid variants (H3, B6, F10, A6, D9) isolated by the screening of the AAV library with the anti-KLH mAb were analyzed by dot blot experiments (FIG. 4). 5.0×10¹⁰ and 1.0×10¹⁰ AAV particles were spotted onto a nitrocellulose membrane using a vacuum device. As negative control wtAAV was spotted ranging from 1.0×10¹⁰ to 1.6×10⁸ capsids per dot. Likewise serial dilutions of BSA (1.0 μg-0.03 μg) were spotted on the membrane as a negative control. As a positive control different dilutions of KLH protein were spotted (1.0 μg-0.02 μg). After blocking of the membrane with blocking buffer (5% milk powder in PBS containing 0.05% Tween-20), the membrane was incubated with the anti-KLH antibody (0.5 μg/ml in 1% milk powder in PBS containing 0.05% Tween-20) used for the screening of the AAV library at 4° C. for 18 h-24 h. After washing of the membrane with PBS/0.05% Tween-20, binding of the anti-KLH antibody to the spotted AAV variants was detected with an anti-mouse IgG (γ) HRP conjugate (CALTAG). The membrane was incubated with the anti-mouse IgG (γ) HRP conjugate for 1 h at room temperature. After washing, signals were detected by chemiluminescence using the ECL system (Amersham Bioscience) (FIG. 4A).

To demonstrate that equal amounts of AAV variants were spotted on the membrane, the AAV capsids were detected using the AAV Capsid-specific mAb A20 (Progen). After stripping of the membrane with stripping buffer (0.1 M glycine, pH 2.5), binding of AAV variants to the membrane was demonstrated using A20 mAb at 5.0 μg/ml in 1% milk powder in PBS containing 0.05% Tween-20. The membrane was incubated with the A20 antibody (Progen) (hybridoma supernatant 1:10 diluted in 1% milk powder in PBS containing 0.05% Tween-20) for 2 h at room temperature. After washing of the membrane with PBS/0.05% Tween-20, binding of the A20 mAb to the spotted AAV variants was detected with an anti-mouse IgG (γ) HRP conjugate (CALTAG). The membrane was incubated with the anti-mouse IgG (γ) HRP conjugate for 1 h at room temperature. After washing, signals were detected by chemiluminescence using the ECL system (Amersham Bioscience) (FIG. 4B).

The result demonstrates that there is a specific detection of AAV capsid variants H3, B6, A6 and B9 by the anti-KLH antibody, which was used for screening of the AAV library. There is no cross-reaction with wtAAV. The weak detection of B6 by the A20 antibody might be due to the immobilization of a lower amount of capsids or due to a poor detection of the B6 variant by the A20 antibody caused by structural modifications of the AAV capsid variant. The weak detection of KLH by A20 in the upper row of panel B is due to incomplete stripping of the membrane shown on the left.

To analyze whether the anti-KLH antibody recognized a structural motif or a linear motif of the AAV variants, 1×10¹⁰ native or heat-inactivated (10 min at 95° C.) capsids were spotted onto a nitrocellulose membrane (FIG. 5). As negative control wtAAV was spotted ranging from 5.0×10¹⁰ to 1.6×10⁹ capsids per dot. As a positive control different dilutions of KLH protein were spotted (1.0 μg-0.03 μg). After blocking, the membrane was incubated with the anti-KLH antibody used for the screening of the AAV library as described above. Binding of the anti-KLH antibody to the spotted AAVLP variants was detected with an anti-mouse IgG HRP conjugate (FIG. 5).

These data demonstrate that native but not heat-denatured H3 and B6 variants are recognized by the anti-KLH antibody, indicating that the antibody recognizes a structural rather than a linear epitope within the AAV capsid. A6 and D9 are not recognized by the antibody most probably due to the low number of spotted capsids (1×10¹⁰).

ELISA Experiments

To confirm the results of the dot blot experiments, the detection of the AAV variants by the KLH antibody was also analyzed in an ELISA format (FIG. 6). 5×10¹⁰ AAV particles (H3, F10, B6, A6, D9) were coated onto a Maxisorp microtiter plate (Nunc). As negative control wtAAV was coated ranging from 5.0×10¹⁰ to 7.8×10⁸ capsids per well. After blocking, the wells were incubated with the anti-KLH antibody used for screening of the AAV library. Binding of the anti-KLH antibody to the immobilized AAV variants was detected with an anti-mouse IgG HRP conjugate using TMB as substrate. The absorbance was read at 450 nm.

These data demonstrate that variants B6 and A6 are detected in the KLH-specific ELISA, although the sensitivity of the ELISA seems to be lower than the sensitivity of the dot blot. This might be due to the binding of lower amounts of AAV particles to the plate or due to structural changes of the capsids caused by the adsorption to the plastic surface of the plate.

5.2. Anti-Idiotype Selection Using an Anti-IgE Antibody

To proof the concept of selection for an anti-idiotype AAV vaccine, an anti-IgE antibody was used for screening of the AAV capsid library. In this experiment, a AAV library was used, whose geno- and phenotype was coupled by infection at GPC 1000 or unspecific uptake at GPC 100 as described above (2.1 and 2.2).

5.2.1. Binding of AAV to Immobilized Anti-IgE Antibody

A cell culture plate (Ø15 cm, TPP) was coated with 10.0 ml anti-IgE antibody (XOLAIR®) at a concentration of 10 μg/ml in coating buffer (0.8 ml 0.2M NaHCO₃, 1.7 ml 0.2M Na₂CO₃ ad 10 ml H₂O) for 18 h-24 h at 4° C. The anti-IgE antibody coated plate was washed three times with 20 ml D-PBS containing 1% Tween-20 to remove unbound antibody. After washing the coated plate was incubated with 20 ml blocking buffer (5% milk powder in D-PBS containing 1% Tween-20) for 2 h at room temperature to avoid unspecific binding of the AAV particles to the plate. The plate was then incubated with 4×10⁸ genome-containing AAV library particles in a total volume of 10 ml blocking buffer for 2 h at room temperature. The genomic titer of the AAV population was determined by quantitative real-time PCR as described above. After incubation of the anti-IgE antibody coated plate with the AAV library, unbound virus was removed by 20 washes with 20 ml D-PBS/1% Tween-20 followed by four washes with 20 ml D-PBS.

5.2.2. Uptake and Amplification of AAV by HeLa Cells

4.0×10⁶ HeLa cells per plate were seeded onto the AAV particles captured by the anti-IgE mAb. Simultaneously, HeLa cells were infected with Adenovirus Type-2 (AdV2) at an MOI of 5 to induce replication of AAV particles. Infection and cultivation of the HeLa cells was performed in a total volume of 20 ml DMEM containing 10% (v/v) fetal calf serum (FCS) and 1% (v/v) Penicillin/Streptomycin for 24 h at 37° C. and 5% CO₂ in a humidified atmosphere. After 48 h of cultivation, HeLa cells were harvested using a cell scraper and collected by centrifugation (3000 g, 10 min, 4° C.). Cells were washed with 5 ml D-PBS. After centrifugation (3000 g, 10 min, 4° C.) the cell pellet was resuspended in 500 μl lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5). Cells were lyzed by three freeze/thaw cycles using liquid nitrogen and a thermoblock tempered at 37° C.

5.2.3. PCR Amplification and Subcloning of the AAV Library Insertion Site

Total DNA was purified from a 50 μl aliquot of the transduced HeLa cell lysate. The cell lysate was diluted fourfold in PBS and total DNA was prepared using the DNeasy Tissue Kit (Qiagen) according to the instructions of the manufacturer. Total DNA was eluted in 50 μl H₂O. The fragment of the AAV genome containing the library insertion site was amplified by PCR using 5 μl of the total DNA prepared from the cell lysate as template and 20 pmol of the primers

BsiWI back (SEQ ID NO: 92) 5′-TAC CAG CTC CCG TAC GTC CTC GGC-3′ and SnaBI forward (SEQ ID NO: 89) 5′-CGC CAT GCT ACT TAT CTA CG-3′ in a total volume of 50 μl. PCR was performed using the High Fidelity Platinum Pfx Polymerase Kit (Invitrogen). After initial heat denaturation of the DNA template at 95° C. for 3 min, DNA was amplified by 35 PCR cycles (45 sec at 95° C. denaturation, 40 sec at 56° C. annealing, 2 min at 68° C. extension). Amplification was terminated after a final extension step at 68° C. for 10 min. An aliquot of the PCR reaction was analyzed on a 1% TBE agarose gel. The PCR product was purified using the PCR Purification Kit (Qiagen). The PCR product was cloned into the BsiWI/SnaBI site of the vector pRC-Kotin. Electro-competent E. coli XL-1 Blue MRF were transformed with the vectors by electroporation. The plasmids of 100 single clones of the cloning reaction were prepared and the insertion site of the library was sequenced using the primer

4066 back (SEQ ID NO: 86) 5′-ATG TCC GTC CGT GTG TGG-3′.

The obtained nucleotide sequences were translated into protein sequences and the 7mer peptide sequences inserted at position I-587 of AAV2 VP was analyzed. The results are summarized in Table 10. AAV particles containing the same peptide sequence at the library insertion site as AAV particles obtained by screening of the library using an uncoated culture plate (see 5.1) were considered as non-specifically bound particles and were excluded from further analysis.

5.2.4. Second, Third and Fourth Round of Anti-IgE Antibody Screening

The number of genomic particles (genomic AAV titer) contained in the HeLa cell lysate was determined by quantitative real-time PCR (see 2.1.3). For the second, third and fourth round of selection, cell culture plates were coated with anti-IgE antibody (XOLAIR®) as described above. Blocking and washing of the coated plates was performed as describe above. Anti-IgE antibody coated plates were incubated with the volume of HeLa cell lysate (containing the AAV pool of the first, second or third selection round, respectively) corresponding to GPC 100 in a total volume of 10 ml blocking buffer. After incubation of the anti-IgE antibody coated plates with the AAV pool obtained from preceding round of selection for 2 h at room temperature, unbound virus was removed by 20 washes with 20 ml D-PBS/1% Tween-20 followed by four washes with 20 ml D-PBS. Uptake and amplification of the anti-IgE mAb bound AAV by HeLa cells was performed as described above. Preparation of total DNA, PCR amplification and subcloning of the AAV library insertion site was performed as described above. The results of the 2nd, 3rd and 4th selection round are summarized in Table 10. AAV particles containing the same peptide sequence at the library insertion site as AAV particles obtained by screening of the library using an uncoated culture plate (see 4.1) were considered as non-specifically bound particles and were excluded from further analysis.

5.2.5. Characterization of AAV Particles Obtained by Anti-IgE Antibody Screening of the AAV Library

AAV particles of the library screening approach were produced and purified as described above. AAV capsid titers were analyzed using the AAV Titration ELISA.

Dot Blot Analysis

The AAV capsid variants (H5, D5, E8, A9, C7, G8) isolated by the screening of the AAV library with the anti-IgE antibody (XOLAIR®) were analyzed by dot blot experiments (FIG. 7). 5.0×10¹° and 1.0×10¹⁰ capsids of the AAV variants were spotted onto a nitrocellulose membrane using a vacuum device except for C7 where 1×10¹⁰ capsids only were spotted. As negative control wtAAV was spotted ranging from 5.0×10¹⁰ to 3.9×10⁸ capsids per dot. Likewise, serial dilutions of BSA (1.0 μg-0.03 μg) were spotted on the membrane as a negative control. As a positive control different dilutions of human IgE protein were spotted (1.0 μg-0.02 μg). After blocking of the membrane with blocking buffer (5% milk powder in PBS containing 0.1% Tween-20), the membrane was incubated with the XOLAIR® antibody (0.15 μg/ml in 1% milk powder in PBS containing 0.05% Tween-20) used for the screening of the AAV library at 4° C. for 18 h-24 h. After washing of the membrane with PBS/0.05% Tween-20, binding of the anti-IgE antibody to the spotted AAV variants was detected with an anti-human IgG HRP conjugate (FIG. 7A)).

To demonstrate that equal amounts of AAV variants were spotted on the membrane, the membrane was stripped as described above and spotted AAV capsids were detected using A20 (FIG. 7B). For this, the membrane was incubated with the A20 antibody (Progen) (hybridoma supernatant 1:10 diluted in 1% milk powder in PBS containing 0.05% Tween-20) for 2 h at room temperature. After washing of the membrane with PBS/0.05% Tween-20, binding of the A20 mAb to the spotted AAV variants was detected with an anti-mouse IgG (γ) HRP conjugate (CALTAG). The result demonstrates that there is a specific detection of AAV variants H5, D5 and E8 by the anti-IgE antibody (XOLAIR®) used for screening of the AAV capsid library.

To demonstrate the specificity of the binding of anti-IgE antibody to the AAV variants, the experiments were repeated and a control mAb (anti-KLH) was included into the experiments (FIG. 8). 5×10¹⁰ and 1×10¹⁰ particles of the AAV variants (H5 only 1×10¹⁰) were dotted onto a nitrocellulose membrane. As negative control wtAAV was spotted ranging from 5.0×10¹⁰ to 1.6×10⁹ capsids per dot. As a positive control different dilutions of human IgE or KLH protein (1.0 μg-0.03 μg) were dotted. After blocking of the membrane with blocking buffer (5% milk powder in PBS containing 0.1% Tween-20), the membrane was incubated with the XOLAIR® antibody (0.15 μg/ml in 1% milk powder in PBS containing 0.05% Tween-20) used for the screening of the AAV library (FIG. 8A) or the control anti-KLH mAb (0.5 μg/ml in 1% milk powder in PBS containing 0.05% Tween-20) (FIG. 8B) at 4° C. for 18 h-24 h. After washing of the membrane with PBS/0.05% Tween-20, binding of the anti-IgE antibody or anti-KLH mAb to the spotted AAV variants was detected with a secondary HRP conjugated antibody. Please note that the signal for D5 (5×10¹⁰ particles) was so strong that the substrate was exhausted at the time of exposure.

These data demonstrate that variants H5, E8 and D5 specifically bind to the anti-IgE antibody, whereas there is no binding to the control anti-KLH antibody. In contrast variant G8 seems to bind to immunoglobulins in an unspecific way.

5.3. Anti-Idiotype Selection Using an Anti-CETP Antibody

To proof the concept of selection for an anti-idiotype AAV vaccine, an anti-CETP antibody was used for screening of the AAV capsid library. In this experiment, a AAV library was used, whose geno- and phenotype was coupled by infection at GPC 1000 described above (2.2).

5.3.1. Binding of AAV to Immobilized Anti-CETP Antibody

A cell culture plate (Ø10 cm, TPP) was coated with 5.0 ml anti-CETP antibody (clone ATM192, Acris-Antibodies) at a concentration of 10 μg/ml in coating buffer (0.8 ml 0.2M NaHCO₃, 1.7 ml 0.2M Na₂CO₃ ad 10 ml H₂O) for 18 h-24 h at 4° C. The anti-CETP antibody-coated plate was washed three times with 10 ml D-PBS containing 1% Tween-20 to remove unbound antibody. After washing the coated plate was incubated with 10 ml blocking buffer (5% milk powder in D-PBS containing 1% Tween-20) for 2 h at room temperature to avoid unspecific binding of the AAV particles to the plate. The plate was then incubated with 1×10⁸ genome-containing AAV library particles in a total volume of 5 ml blocking buffer for 2 h at room temperature. The genomic titer of the AAV population was determined by quantitative real-time PCR as described above. After incubation of the anti-CETP antibody-coated plate with the AAV library, unbound virus was removed by 20 washes with 10 ml D-PBS/1% Tween-20 followed by four washes with 10 ml D-PBS.

5.3.2. Uptake and Amplification of AAV by HeLa Cells

1.0×10⁶ HeLa cells per plate were seeded onto the AAV particles captured by the anti-CETP mAb. Simultaneously, HeLa cells were infected with AdV2 at an MOI of 5 to induce replication of AAV particles. Infection and cultivation of the HeLa cells was performed in a total volume of 10 ml DMEM containing 10% (v/v) fetal calf serum (FCS) and 1% (v/v) Penicillin/Streptomycin for 48 h at 37° C. and 5% CO₂ in a humidified atmosphere. After 48 h of cultivation, HeLa cells were harvested using a cell scraper and collected by centrifugation (3000 g, 10 min, 4° C.). Cells were washed with 5 ml D-PBS. After centrifugation (3000 g, 10 min, 4° C.) the cell pellet was resuspended in 250 μl lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5). Cells were lyzed by three freeze/thaw cycles using liquid nitrogen and a thermoblock tempered at 37° C.

5.3.3. PCR Amplification and Subcloning of the AAV Library Insertion Site

Total DNA was purified from a 50 μl aliquot of the transduced HeLa cell lysate. The cell lysate was diluted fourfold in PBS and total DNA was prepared using the DNeasy Tissue Kit according to the instructions of the manufacturer. Total DNA was eluted in 50 μl H₂O. The fragment of the AAV genome containing the library insertion site was amplified by PCR using 5 μl of the total DNA prepared from the cell lysate as template and 20 pmol of the primers

BsiWI back (SEQ ID NO: 88) 5′-TAC CAG CTC CCG TAC GTC CTC GGC-3′ and SnaBI forward (SEQ ID NO: 89) 5′-CGC CAT GCT ACT TAT CTA CG-3′ in a total volume of 50 μl. PCR was performed using the High Fidelity Platinum Pfx Polymerase Kit (Invitrogen). After initial heat denaturation of the DNA template at 95° C. for 3 min, DNA was amplified by 35 PCR cycles (45 sec at 95° C. denaturation, 40 sec at 56° C. annealing, 2 min at 68° C. extension). Amplification was terminated after a final extension step at 68° C. for 10 min. An aliquot of the PCR reaction was analyzed on a 1% TBE agarose gel. The PCR product was purified using the PCR Purification Kit (Qiagen). The PCR product was cloned into the BsiWI/SnaBI site of the vector pRC-Kotin. Electro-competent E. coli XL-1 Blue MRF were transformed with the vectors by electroporation. The plasmids of 100 single clones of the cloning reaction were prepared and the insertion site of the library was sequenced using the primer

4066 back (SEQ ID NO: 86) 5′-ATG TCC GTC CGT GTG TGG-3′.

The obtained nucleotide sequences were translated into protein sequences and the 7mer peptide sequence inserted at position I-587 of AAV2 VP was analyzed. The results are summarized in Table 10. AAV particles containing the same peptide sequence at the library insertion site as AAV particles obtained by screening of the library using an uncoated culture plate (see 4.1) were considered as non-specifically bound particles and were excluded from further analysis

5.3.4. Second and Third Round of Anti-CETP Antibody Screening

The number of genomic particles (genomic AAV titer) contained in the HeLa cell lysate was determined by quantitative real-time PCR (see 2.1.3). For the second and third round of selection, cell culture plates were coated with anti-CETP antibody as described above. Blocking and washing of the coated plates was performed as described above. Anti-CETP antibody-coated plates were incubated with the volume of HeLa cell lysate (containing the AAV pool of the first and second selection round, respectively) corresponding to GPC 100 in a total volume of 5 ml blocking buffer. After incubation of the anti-CETP antibody coated plates with the AAV pool obtained from the first and second round of selection for 2 h at room temperature, unbound virus was removed by 20 washes with 10 ml D-PBS/1% Tween-20 followed by four washes with 10 ml D-PBS. Uptake and amplification of the anti-CETP mAb-bound AAV by HeLa cells was performed as described above. Preparation of total DNA, PCR amplification and subcloning of the AAV library insertion site was performed as described above. AAV particles containing the same peptide sequence at the library insertion site as AAV particles obtained by screening of the library using an uncoated culture plate (see 4.1) were considered as non-specifically bound particles and were excluded from further analysis.

5.3.5. Characterization of AAV Particles Obtained by Anti-CETP Antibody Screening of the AAV Library

AAV particles of the library screening approach were produced and purified as described above. AAV capsid titers were analyzed using the AAV Titration ELISA.

Dot Blot Analysis

The AAV capsid variants B8 and C4 isolated by the screening of the AAV library with the anti-CETP antibody were analyzed by dot blot experiments (FIG. 9). 5.0×10¹⁰ and 1.0×10¹⁰ AAV variants were spotted onto a nitrocellulose membrane using a vacuum device. As negative control wtAAV was spotted ranging from 5.0×10¹⁰ to 3.2×10⁹ capsids per dot. Likewise, serial dilutions of BSA (1.0 μg-0.03 μg) were spotted on the membrane as a negative control. After blocking of the membrane with blocking buffer (5% milk powder in PBS containing 0.1% Tween-20), the membrane was incubated with the anti-CETP antibody (5 μg/ml in 1% milk powder in PBS containing 0.05% Tween-20) used for the screening of the AAV library at 4° C. for 18 h-24 h. After washing of the membrane with PBS/0.05% Tween-20, binding of the anti-CETP antibody to the spotted AAV variants was detected with an anti-mouse IgG HRP conjugate. The membrane was incubated with an anti-mouse IgG HRP conjugate for 1 h at room temperature. After washing, signals were detected by chemiluminescence using the ECL system (Amersham Bioscience) (FIG. 9A).

To demonstrate that equal amounts of AAV variants were spotted on the membrane, the membrane was stripped as described above and spotted AAV capsids were detected using A20 (FIG. 9B). For this, the membrane was incubated with the A20 antibody (Progen) (1:10 diluted in 1% milk powder in PBS containing 0.05% Tween-20) for 2 h at room temperature. After washing of the membrane with PBS/0.05% Tween-20, binding of the A20 mAb to the spotted AAV variants was detected with an anti-mouse IgG (γ) HRP conjugate (CALTAG). The membrane was incubated with the anti-mouse IgG (γ) HRP conjugate for 1 h at room temperature. After washing, signals were detected by chemiluminescence using the ECL system (Amersham Bioscience) (FIG. 9B). The result demonstrates that there is a specific detection of AAV variants B8 and C4 by the anti-CETP antibody used for screening of the AAV capsid library.

5.4. Optimizing the Presentation of the Selection Antibody

The presentation of the antibody used for selection can be improved by pre-coating of the cell culture plates or other supports (like sepharose beads) with a species and isotype-specific F(ab)₂ fragment that binds to the constant F_(c) region of the selection antibody. This allows an orientated presentation of the selection antibody with the constant region bound to the immobilized F(ab)₂ fragment and leaves the idiotype portion of the antibody accessible for AAV variants. Therefore, a lower number of false-positive AAV variants that bind to other regions of the selection antibody (e.g. F_(c) portion) will be isolated in the screening approach. Likewise other molecules, including protein A or protein G, that bind to the constant region of immunoglobulins can be used to orient the selection antibody. In addition, the surface density of immobilized selection antibodies can be increased by the use of other supports (like sepharose beads) instead of plastic cell culture plates.

5.5. PCR-Based Amplification of the Genome of AAV Particles Captured by a Selection Antibody

As an alternative to cellular uptake and amplification of AAV particles following infection of HeLa cells by AdV (as described above), the genome of AAV particles bound to a target antibody after the first or a subsequent selection round can be amplified by a PCR-based approach. AAV particles captured by the selection antibody are lyzed by a suitable buffer and DNA is isolated by a suitable method. For example, the AAV genome can be isolated using the DNeasy Blood & Tissue Kit (Qiagen) according to the protocol “Purification of Total DNA from Animal Blood or Cells” provided by the manufacturer. The fragment of the cap-gene containing the library insertion site with the respective inserted sequence can be amplified by PCR using the isolated DNA and suitable primers. The fragment can be subcloned into a suitable vector and analyzed by sequencing. For example, the DNA fragment of the cap gene containing the library insertion site can be amplified by Platinum Pfx DNA polymerase (Invitrogen) using a PCR. enhancer solution (Invitrogen), Pfx amplification buffer (Invitrogen) and the primers

BsiWI-back: (SEQ ID NO: 88) 5′-TAC CAG CTC CCG TAC GTC CTC GGC-3′ and SnaBI-forward: (SEQ ID NO: 89) 5′-CGC CAT GCT ACT TAT CTA CG-3′ according to the following PCR program: Initial denaturation at 95° C., 3 min; 35 amplification cycles: 95° C. for 45 s, 56° C. for 40 s, 68° C. for 2 min; and a final Elongation at 68° C., 10 min.

Following restriction with BsiWI and SnaBI, the PCR product can be cloned into the BsiwI/SnaBI linearized vector pUCAV2 (pUCAV2 is described in detail in U.S. Pat. No. 6,846,665). Clones can be analyzed by sequencing using the primer

4066back (SEQ ID NO: 86) 5′-ATG TCC GTC CGT GTG TGG-3′

6. Generation of Modified AAV Variants by Insertion of Epi- or Mimotope Sequences at Position I-587 or I-453 of the AAV Capsid by Genetic Manipulation

The approach described below is used for the insertion of epi- or mimotopes into the AAV capsid at position I-587 using a defined cloning strategy. This strategy includes the generation of a NotI and AscI restriction site within the cap gene by site-directed mutagenesis that allows the insertion of DNA fragments encoding epi- or mimotope at position I-587 of AAV cap flanked by a short or long alanine adaptor sequence.

6.1. Creation of Singular NotI and AscI Restriction Sites in Vector pCI-VP2

The vector pCI-VP2 was created by PCR amplification of the AAV2 VP2 gene mutating the minor ACG start codon into an ATG and cloning of the respective PCR product into the polylinker sequence of pCI (Promega). The NotI site at nucleotide 18 of pCI-VP2 (nucleotide 1099 of pCI) was destroyed by site directed mutagenesis using the primers

mutashe-3: (SEQ ID NO: 111) 5′-GAG TCG ACC CGG GCA GCC GCT TCG AGC-3′ and mutashe-4 (SEQ ID NO: 112) 5′-GCT CGA AGC GGC TGC CCG GGT CGA CTC-3′ together with the QuickChange II Site-Directed Mutagenesis Kit (Stratagene) according to the instructions of the manufacturer. The resulting vector was referred to as pCI-VP2-ΔNot18. To introduce a NotI and AscI restriction site that allows the cloning of epitope or mimotope sequences at position I-587 of the AAV capsid, the vector pCI-VP2-ΔNot18 was modified by site directed mutagenesis using the primers

pCI-VP2-ΔNot-I587-for (SEQ ID NO: 113) 5′-CC AAC CTC CAG AGA GGC AAC GCG GCC GCA AGG CGC GCC AAG CAG CTA CCG CAG-3′ and pCI-VP2-ΔNot-I587-rev (SEQ ID NO: 114) 5′-CTG CGG TAG CTG CTT GGC GCG CC TT GCG GCC GCG TTG CCT CTC TGG AGG TTG G-3′.

Site specific mutagenesis was performed using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene) according to the instructions of the manufacturer. The resulting vector is referred to as pCIVP2-I587-NotI-AscI.

6.2. Cloning of Epitope or Mimotope Sequences into pCIVP2-I587-NotI-AscI

For cloning of epi- or mimotope sequences into pCIVP2-I587-NotI-AscI sense and anti-sense oligonucleotides were designed that encode the respective epi- or mimotope sequences with a short or long alanine adaptor sequence and contain a 5″-site extension. The 5″-site extension of the oligonucleotides was designed so that annealing of the sense and anti-sense oligonucleotides results in a dsDNA with 5″-site and 3″-site overhangs compatible with overhangs generated by NotI and AscI restriction of the plasmid pCIVP2-I587-NotI-AscI. The sequences of the oligonucleotides and the respective epi- or mimotope sequences including the alanine adaptors are summarized in Table 11. Each of the inserted epi- or mimotope sequences is flanked by a short or long alanine adaptor according to the following scheme (X_(n) represents the mimotope or epitope sequence):

-   -   short Ala adaptor: (Ala)₃-X_(n)-(Ala)₂     -   long Ala adaptor: (Ala)₅-X_(n)-(Ala)₅

TABLE 11 Oligonucleotides used for cloning of epi- or mimotope sequences Name/ sense anti-sense Alanine Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor Kricek Epitope 5′ GGCCGCAGTGAACC 5′ CGCGCCGCGCCGGAG short VNLTWSRASG TGACCTGGAGCAGAGCC GCTCTGCTCCAGGTCAGG (SEQ ID NO: 85) TCCGGCGCGG 3′ TTCACTGC 3′ SEQ ID NO: 115 SEQ ID NO: 116 5′ GGCCGCAGCGGCGG 5′ CGCGCCGCCGCCGCC long TGAACCTGACCTGGAGC GCGCCGGAGGCTCTGCTC AGAGCCTCCGGCGCGGC CAGGTCAGGTTCACCGCC GGCGGCGG 3′ GCTGC 3′ SEQ ID NO: 117 SEQ ID NO: 118 Rudolf Mimotope 5′ GGCCGCAGAATTCT 5′ CGCGCCGCGTCTCCG short EFCINHRGYWVCGD GCATAAACCACAGGGGA CACACCCAGTATCCCCTG (SEQ ID NO: 84) TACTGGGTGTGCGGAGA TGGTTTATGCAGAATTCT CGCGG 3′ GC 3′ SEQ ID NO: 119 SEQ ID NO: 120 5′ GGCCGCAGCGGCGG 5′ CGCGCCGCCGCCGCC long AATTCTGCATAAACCAC GCGTCTCCGCACACCCAG AGGGGATACTGGGTGTG TATCCCCTGTGGTTTATG CGGAGACGCGGCGGCGG CAGAATTCCGCCGCTGC CGG 3′ 3′ SEQ ID NO: 121 SEQ ID NO: 122 CETP-intern Epitope 5′ GGCCGCATGCGACG 5′ CGCGCCGCGTCTGGT short CDAGSVRTNAPD CTGGCAGTGTGCGCACC GCATTGGTGCGCACACTG SEQ ID NO: 123 AATGCACCAGACGCGG CCAGCGTCGCATGC 3′ 3′ SEQ ID NO: 125 SEQ ID NO: 124 5′ GGCCGCAGCGGCGT 5′ CGCGCCGCCGCCGCC long GCGACGCTGGCAGTGTG GCGTCTGGTGCATTGGTG CGCACCAATGCACCAGA CGCACACTGCCAGCGTCG CGCGGCGGCGGCGG 3′ CACGCCGCTGC 3′ SEQ ID NO: 126 SEQ ID NO: 127

To anneal the oligonucleotides 50.0 μg of the sense oligonucleotide and 50.0 μg of the anti-sense oligonucleotide were mixed in a total volume of 200 μl 1×PCR-Buffer (Qiagen) and incubated for 3 min at 95° C. in a thermomixer. After 3 min at 95° C. the thermomixer was switched off and the tubes were left in the incubator for an additional 2 h to allow annealing of the oligonucleotides during the cooling down of the incubator. To clone the annealed oligonucleotides into pCIVP2-I587-NotI-AscI the vector was linearized by restriction with NotI and AscI and the cloning reaction was performed using the Rapid DNA Ligation Kit (Roche). Briefly, the annealed oligonucleotides were diluted 10-fold in 1×DNA Dilution Buffer and incubated for 5 min at 50° C. 100 ng of these annealed oligonucleotides and 50 ng of the linearized vector pCIVP2-I587-NotI-AscI were used in the ligation reaction, which was performed according to the instructions of the manufacturer of the Rapid DNA Ligation Kit (Roche). E. coli XL1 blue were transformed with an aliquot of the ligation reaction and plated on LB-Amp agar plates. Plasmids were prepared according to standard procedures and were analyzed by sequencing.

6.3. Subcloning of Epitope or Mimotope Sequences Form pCIVP2 into pUCAV2

For production of recombinant AAV particles carrying a mimo- or epitope insertion at position I-587 the BsiWI/XmaI fragment of pCI-VP2-587-NotI-AscI encoding a VP2 fragment containing the epitope or mimotope at position I-587 was sub-cloned into pUCAV2, which was modified as described below.

Cloning of vector pUCAV2 is described in detail in U.S. Pat. No. 6,846,665. Basically, this vector contains the complete AAV genome (BgI II fragment) derived from pAV2 (Laughlin et al., 1983) cloned into BamHI of pUC19.

pUCAV2 is used for production of the modified AAV particles. Since there are three XmaI sites in pUCAV2 it is not possible to use the XmaI site of pUCAV2 for subcloning of the BsiWI/XmaI fragment of pCI-VP2-587-NotI-AscI. Therefore, a new AgeI site was introduced into pUCAV2 that is compatible with XmaI and is not present in pUCAV2. To introduce the AgeI site pUCAV2 was linearized by SnaBI (position nt 2873 of pUCAV2), dephosphorylated and subsequently blunt-end ligated with a short ds oligonucleotide adaptor containing an internal AgeI site. The ds oligonucleotide adaptor was generated by annealing of a

sense (SEQ ID NO: 128) 5′-GTA GCC CTG GAA ACT AGA ACC GGT GCC TGC GCC-3′ and anti-sense (SEQ ID NO: 129) 5′-GGC GCA GGC ACC GGT TCT AGT TTC CAG GGC TAC 3′ oligonucleotide containing an AgeI restriction site as described above. The annealed oligonucleotides were ligated with the SnaBI linearized, dephosphorylated pUCAV2 using the Rapid DNA Ligation Kit (Roche) as described above. The resulting vector is referred to as pUCAV2-AgeI. pUCAV2-AgeI was linearized with BsiWI and AgeI and ligated with the BsiWI/XmaI fragment of pCI-VP2-587-NotI-AscI encoding the VP2 fragment containing the respective epitope or mimotope at position I-587.

6.4. Production of AAV Variants by Co-Transfection of HEK 293 T-Cells

For production of AAV variants HEK 293-T cells were co-transfected with the vector plasmid pUCAV2 containing the subcloned mimo- or epiotope sequence, and the helper plasmid pUCAdV as described above (3.2). AAV variants were purified by Iodixanol gradient centrifugation as described above (3.3).

6.4.1. Insertion of a CETP Epitope into the AAV2 Capsid at Position I-587

An epitope (CDAGSVRTNAPD, SEQ ID NO: 123) of rabbit CETP (cholesteryl ester transfer protein) was introduced at position I-587 of AAV2 by the cloning approach described above. The epitope is flanked by a short or long alanine adaptor. For production of AAV variants HEK 293-T cells were co-transfected with the vector plasmid pUCAV2 containing the subcloned CETP epitope sequence at position I-587, and the helper plasmid pUCAdV as described above (4.2). AAV variants were purified by Iodixanol gradient centrifugation as described above (4.3).

The AAV capsid variants AAV-CETP-587-short and AAV-CETP-587-long were analyzed by dot blot experiments (FIG. 10). 5×10¹⁰ purified AAV particles were spotted onto a nitrocellulose membrane using a vacuum device. Likewise, 5×10¹⁰ or 1×10¹⁰ purified AAV particles displaying the same epitope of rabbit CETP flanked by a short or long Ala adaptor sequence at position I-453 of AAV2 (see 6.4.3) were spotted onto the same membrane. As negative control wtAAV was spotted ranging from 5.0×10¹⁰ to 6.3×10⁹ capsids per dot. After blocking of the membrane with blocking buffer (5% milk powder in PBS containing 0.05% Tween-20), the membrane was incubated with a polyclonal anti-CETP serum generated by immunizing rabbits with the CETP epitope coupled to KLH. After washing of the membrane with PBS/0.05% Tween-20, binding of the anti-CETP antibodies to the spotted AAV variants was detected with an anti-rabbit IgG HRP conjugate (CALTAG). After washing, signals were detected by chemiluminescence using the ECL system (Amersham Bioscience).

The result demonstrate that there is a specific detection of the CETP epitope inserted into the AAV capsid at position I-587 and I-453 (for methods see 6.4.3) by the respective CETP antibody demonstrating that the epitope is displayed on the surface of the AAV particle.

6.4.2. Insertion of an IgE Epitope into the AAV2 Capsid at Position I-587

An epitope of IgE (VNLTWSRASG, SEQ ID NO: 85), that is recognized by the monoclonal anti-IgE antibody Bsw17 (Kricek et al., 1999)), was introduced at position I-587 of AAV2 by the cloning approach described above. The epitope is flanked by a long alanine adaptor in the AAV capsid. For production of the respective AAV variant (AAV-Kricek) HEK 293-T cells were co-transfected with the vector plasmid pUCAV2 containing the subcloned IgE epitope sequence at position I-587, and the helper plasmid pUCAdV as described above (3.2). AAV variants (AAV-Kricek) were purified by Iodixanol gradient centrifugation as described above (3.3).

The AAV capsid variant AAV-Kricek was analyzed by a dot blot experiment (FIG. 14). 2.5×10¹⁰ AAV-Kricek particles were dotted onto a nitrocellulose membrane using a vacuum device. As negative control serial dilutions of wtAAV (5.0×10¹⁰ to 1.6×10⁹) or the AAV variant D5 (see 4.2.5.1) were dotted. As a positive control human IgE was dotted ranging from 1.0 μg to 0.03 μg. After blocking of the membrane with blocking buffer (5% milk powder in PBS containing 0.05% Tween-20), the membrane was incubated with the anti-IgE mAb Bsw17. After washing of the membrane with PBS/0.05% Tween-20, binding of Bsw17 to the spotted AAV variants was detected with an anti-mouse IgG HRP conjugate (CALTAG). After washing, signals were detected by chemiluminescence using the ECL system (Amersham Bioscience).

The result shows that there is a specific detection of the AAV-Kricek particles by the Bsw17 mAb demonstrating that the antibody recognizes the IgE derived epitope (VNLTWSRASG, SEQ ID NO: 85) integrated in the AAV capsid at position I-587.

6.4.3. Insertion of a CETP Epitope into the AAV2 Capsid at Position I-453

The approach described below is used for the insertion of a CETP epitope (CDAGSVRTNAPD, SEQ ID NO: 123) into the AAV capsid at position I-453.

Creation of Singular NotI and AscI Restriction Sites in Vector pCI-VP2

The vector pCI-VP2 was created by PCR amplification of the AAV2 VP2 gene mutating the minor ACG start codon into an ATG and cloning of the respective PCR product into the polylinker sequence of pCI (Promega). The NotI site at nucleotide 18 of pCI-VP2 (nucleotide 1099 of pCI) was destroyed as described above (6.1). The resulting vector was referred to as pCI-VP2-ΔNot18. To introduce a NotI and AscI restriction site that allows the cloning of epitope or mimotope sequences at position I-453 of the AAV capsid, the vector pCI-VP2-ΔNot18 was modified by site directed mutagenesis using the primers

mutashe-5 (SEQ ID NO: 130) 5′-CA AAC ACT CCA AGT GGA GGG CGC GCC GCT ACC ACC ACG CAG TC-3′ and mutashe-6 (SEQ ID NO: 131) 5′-GA CTG CGT GGT GGT AGC GGC GCG CCC TCC ACT TGG AGT GTT TG-3′ to introduce the AscI site first as well as the primers

mutashe-7 (SEQ ID NO: 132) 5′-CA AAC ACT CCA AGT GGA GCG GCC GCA GGG CGC GCC GCT AC-3′ and mutashe-8 (SEQ ID NO: 133) 5′-GT AGC GGC GCG CCC TGC GGC CGC TCC ACT TGG AGT GTT TG -3′ to introduce the NotI site subsequently.

Site specific mutagenesis was performed using the QuickChange II Site-Directed Mutagenesis Kit (Stratagene) according to the instructions of the manufacturer. The resulting vector is referred to as pCIVP2-I453-NotI-AscI.

Cloning of CETP Epitope into pCIVP2-I453-NotI-AscI

For cloning of the CETP epitope (CDAGSVRTNAPD, SEQ ID NO: 123) into pCIVP2-I453-NotI-AscI forward and reverse oligonucleotides were designed that encode the respective epitope sequence flanked by a short

(SEQ ID NO: 134) (Ala)₃-CDAGSVRTNAPD-R-(Ala)₂ or long (SEQ ID NO: 135) (Ala)₅-CDAGSVRTNAPD-(Ala)₂-R-(Ala)₂ alanine adaptor sequence and contain 5′-site extensions (Table 12). The 5″-site extension of the oligonucleotides was designed so that annealing of the forward and reverse oligonucleotides results in a dsDNA with 5″-site and 3″-site overhangs compatible with overhangs generated by NotI and AscI restriction of the plasmid pCIVP2-I453-NotI-AscI.

TABLE 12 Oligonucleotides used for cloning of CETP epitope sequence at position I-453 Name/ Forward Reverse Alanine Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor CETP-intern Epitope 5′-ggccgcatgcgacgctgg 5′-cgcggtctggtgcattggt short CDAGSVRTNAPD cagtgtgcgcaccaatgcacc gcgcacactgccagcgtcgc SEQ ID NO: 123 agac-3′ a tgc-3′ SEQ ID NO: 136 SEQ ID NO: 137 5′-ggccgcagccgcatgcga 5′-cgcgtgccgcgtctggtgc long cgctggcagtgtgcgcaccaat attggtgcgcacactgccagc gcaccagacgcggca-3′ gtcgcatgcggctgc-3′ SEQ ID NO: 138 SEQ ID NO: 139

To anneal the oligonucleotides 50.0 μg of the forward oligonucleotide and 50.0 μg of the reverse oligonucleotide were mixed in a total volume of 200 μl 1×PCR-Buffer (Qiagen) and incubated for 3 min at 95° C. in a thermomixer. After 3 min at 95° C. the thermomixer was switched off and the tubes were left in the incubator for an additional 2 h to allow annealing of the oligonucleotides during the cooling down of the incubator. To clone the annealed oligonucleotides into pCIVP2-I453-NotI-AscI the vector was linearized by restriction with NotI and AscI and the cloning reaction was performed using the Rapid DNA Ligation Kit (Roche). Briefly, the annealed oligonucleotides were diluted 10-fold in 1×DNA Dilution Buffer and incubated for 5 min at 50° C. 100 ng of these annealed oligonucleotides and 50 ng of the linearized vector pCIVP2-I453-NotI-AscI were used in the ligation reaction, which was performed according to the instructions of the manufacturer of the Rapid DNA Ligation Kit (Roche). E. coli XL1 blue were transformed with an aliquot of the ligation reaction and plated on LB-Amp agar plates. Plasmids were prepared according to standard procedures and were analyzed by sequencing.

Subcloning of the CETP Epitope from pCIVP2 into pUCAV2 at Position I-453

For production of recombinant AAV particles carrying the CETP epitope at position I-453 the BsiWI/XmaI fragment of pCI-VP2-453-NotI-AscI encoding a VP2 fragment containing the epitope at position I-453 was sub-cloned into pUCAV2, which was modified as described above (6.3). pUCAV2-AgeI was linearized with BsiWI and AgeI and ligated with the BsiWI/XmaI fragment of pCI-VP2-453-NotI-AscI encoding the VP2 fragment containing the CETP epitope at position I-453.

Production of AAV Variants by Co-Transfection of HEK 293 T-Cells

For production of AAV variants HEK 293-T cells were co-transfected with the vector plasmid pUCAV2 containing the subcloned CETP epitope sequence at position I-453, and the helper plasmid pUCAdV as described above (4.2). AAV variants were purified by Iodixanol gradient centrifugation as described above (4.3).

The AAV capsid variants AAV-CETP-453-short and AAV-CETP-453-long were analyzed by dot blot experiments as described above (6.4.1, FIG. 10).

6.4.4. Generation of Further AAV Variants

Insertion of CETP Epitopes into the AAV2 Capsid at Position I-587

The following rabbit CETP derived were cloned into position I-587 of the AAV2 capsid using annealed oligonucleotides as described above and were used for production of AAV particles. Each of the inserted epitope sequences is flanked by one of the following alanine/glycine adaptors according to the following scheme (X_(n) represents the epitope sequence):

-   -   Type I adaptor: Ala-(Gly)₃-X_(n)-(Gly)₄-Ala     -   Type II adaptor: Ala-(Gly)₄-X_(n)-(Gly)₄-Ala     -   Type III adaptor: Ala-(Gly)₄-X_(n)-Ala-(Gly)₃-Ala

TABLE 13 CETP derived epitopes. Name/ sense anti-sense Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor CETP TP10 Epitope 5′GGCCGGCGGAGGTGCCAA 5′CGCGCACCGCCACC Type I AKAVSNLTESRS GGCCGTGAGCAACCTACCGA CCCGCTCTGCAGGCTC ESLQS GAGCAGAAGCGAGAGCCTGC TCGCTTCTGCTCTCGG SEQ ID NO: 237 AGAGCGGGGGTGGCGGTG TCAGGTTGCTCACGGC 3′ CTTGGCACCTCCGCC SEQ ID NO: 245 3′ SEQ ID NO: 246 CETP TP11 Epitope 5′GGCCGGCGGAGGTAGCCT 5′CGCGCACCGCCACC Type I SLTGDEFKKVLET GACCGGCGACGAATTCAAGA CCCGGTCTCCAGCACC SEQ ID NO: 238 AGGTGCTGGAGACCGGGGGT TTCTTGAATTCGTCGC GGCGGTG 3′ CGGTCAGGCTACCTCC SEQ ID NO: 247 GCC 3′ SEQ ID NO: 248 CETP TP12 Epitope 5′GGCCGGCGGAGGTAGAGA 5′CGCGCACCGCCACC Type I REAVAYRFEED GGCCGTGGCCTACAGATTCG CCCGTCCTCTTCGAAT SEQ ID NO: 239 AAGAGGACGGGGGTGGCGGT CTGTAGGCCACGGCCT G 3′ CTCTACCTCCGCC 3′ SEQ ID NO: 249 SEQ ID NO: 250 CETP TP13 Epitope 5′GGCCGGCGGAGGTATCAA 5′CGCGCACCGCCACC Type I INPEIITLDG CCCCGAGATCATCACCCTGG CCCGCCGTCCAGGGTG SEQ ID NO: 240 ACGGCGGGGGTGGCGGTG ATGATCTCGGGGTTGA 3′ TACCTCCGCC 3′ SEQ ID NO: 251 SEQ ID NO: 252 CETP TP18 Epitope 5′GGCCGGCGGAGGTGACAT 5′CGCGCACCGCCACC Type I DISVTGAPVITAT CAGCGTGACCGGTGCACCCG CCCCAGGTAGGTGGCG YL TGATCACCGCCACCTACCTG GTGATCACGGGTGCAC SEQ ID NO: 241 GGGGGTGGCGGTG 3′ CGGTCACGCTGATGTC SEQ ID NO: 253 ACCTCCGCC 3′ SEQ ID NO: 254 CETP TP20 Epitope 5′GGCCGGCGGAGGTGACAT 5′CGCGCACCGCCACC Type I DISVTGAPVITA CAGCGTGACCGGTGCACCCG CCCGGCGGTGATCACG SEQ ID NO: 242 TGATCACCGCCGGGGGTGGC GGTGCACCGGTCACGC GGTG 3′ TGATGTCACCTCCGCC SEQ ID NO: 255 3′ SEQ ID NO: 256 Ritsch-1 Epitope 5′GGCCGGCGGAGGTGACCA 5′CGCGCACCGCCACC Type I DQSVDFEIDSA GAGCGTGGACTTCGAGATCG CCCGGCGCTGTCGATC SEQ ID NO: 243 ACAGCGCCGGGGGTGGCGGT TCGAAGTCCACGCTCT G 3′ GGTCACCTCCGCC 3′ SEQ ID NO: 257 SEQ ID NO: 258 Ritsch-3 Epitope 5′GGCCGGCGGAGGTAAGAA 5′CGCGCACCGCCACC Type I KNVSEAFPLRA CGTGAGCGAGGCCTTCCCTC CCCGGCTCTCAGAGGG SEQ ID NO: 244 TGAGAGCCGGGGGTGGCGGT AAGGCCTCGCTCACGT G 3′ TCTTACCTCCGCC 3′ SEQ ID NO: 259 SEQ ID NO: 260

The following sequences, which are human homologues to the corresponding rabbit CETP sequences can be integrated into the AAV2 capsid at position I-587 according to the methods described above:

TABLE 14 CETP derived epitopes at position I-587 Epitope Rabbit Sequence Human Sequence CETP CDAGSVRTNAPD CDSGRVRTDAPD intern SEQ ID NO: 123 SEQ ID NO: 223 CETP FPKHLLVDFLQSLS FPEHLLVDFLQSLS C-Term SEQ ID NO: 261 SEQ ID NO: 224 TP10 AKAVSNLTESRSESLQS PKTVSNLTESSSESVQS SEQ ID NO: 237 SEQ ID NO: 214 TP11 SLTGDEFKKVLET SLMGDEFKAVLET SEQ ID NO: 238 SEQ ID NO: 215 TP12 REAVAYRFEED QHSVAYTFEED SEQ ID NO: 239 SEQ ID NO: 216 TP13 INPEIITLDG INPEIITRDG SEQ ID NO: 240 SEQ ID NO: 217 TP18 DISVTGAPVITATYL DISLTGDPVITASYL SEQ ID NO: 241 SEQ ID NO: 218 TP20 DISVTGAPVITA DISLTGDPVITA SEQ ID NO: 242 SEQ ID NO: 219 Ritsch-1 DQSVDFEIDSA DQSIDFEIDSA SEQ ID NO: 243 SEQ ID NO: 220 Ritsch-2 KNVSEAFPLRAFPPGLLGDS KNVSEDLPLPTFSPTLLGDS SEQ ID NO: 262 SEQ ID NO: 221 Ritsch-3 KNVSEAFPLRA KNVSEDLPLPT SEQ ID NO: 244 SEQ ID NO: 222 Insertion of CETP Epitopes into the AAV2 Capsid at Position I-453

The following rabbit CETP derived epitopes were cloned into position I-453 of the AAV2 capsid using annealed oligonucleotides as described above. Each of the inserted epitope sequences in the AAV2 backbone at I-453 is flanked by the following alanine/glycine adaptors according to the following scheme (X_(n) represents the epitope sequence):

-   -   Type I Ala/Gly adaptor: (Ala)₂-(Gly)₃-X_(n)-(Gly)₄-Arg-(Ala)₂     -   Type II Ala/Gly adaptor: (Ala)₃-(Gly)₃-X_(n)-(Gly)₄-Arg-(Ala)₂

TABLE 15 rabbit CETP derived epitopes at position I-453 Name/ sense anti-sense Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor CETP TP10 Epitope 5′GGCCGGCGGTGGAGCCA 5′CGCGTCCACCGCCACCG Type I AKAVSNLTESRS AGGCCGTGAGCAACCTGAC CTCTGCAGGCTCTCGCTTC Ala/Gly ESLQS CGAGAGCAGAAGCGAGAGC TGCTCTCGGTCAGGTTGCT SEQ ID NO: 237 CTGCAGAGCGGTGGCGGTG CACGGCCTTGGCTCCACCG GA 3′ CC 3′ SEQ ID NO: 263 SEQ ID NO: 264 CETP TP11 Epitope 5′GGCCGGCGGTGGAAGCC 5′CGCGTCCACCGCCACCG Type I SLTGDEFKKVLET TGACCGGCGACGAATTCAA GTCTCCAGCACCTTCTTGA Ala/Gly SEQ ID NO: 238 GAAGGTGCTGGAGACCGGT ATTCGTCGCCGGTCAGGCT GGCGGTGGA 3′ TCCACCGCC 3′ SEQ ID NO: 265 SEQ ID NO: 266 CETP TP12 Epitope 5′GGCCGGCGGTGGAAGAG 5′CGCGTCCACCGCCACCG Type I REAVAYRFEED AGGCCGTGGCCTACAGATT TCCTCTTCGAATCTGTAGG Ala/Gly SEQ ID NO: 239 CGAAGAGGACGGTGGCGGT CCACGGCCTCTCTTCCACC GGA 3′ GCC 3′ SEQ ID NO: 267 SEQ ID NO: 268 CETP TP13 Epitope 5′GGCCGGCGGTGGAATCA 5′CGCGTCCACCGCCACCG Type I INPEIITLDG ACCCCGAGATCATCACCCT CCGTCCAGGGTGATGATCT Ala/Gly SEQ ID NO: 240 GGACGGCGGTGGCGGTGGA CGGGGTTGATTCCACCGCC 3′ 3′ SEQ ID NO: 269 SEQ ID NO: 270 CETP TP18 Epitope 5′GGCCGGCGGTGGAGACA 5′CGCGTCCACCGCCACCC Type I DISVTGAPVITAT TCAGCGTGACCGGTGCACC AGGTAGGTGGCGGTGATCA Ala/Gly YL CGTGATCACCGCCACCTAC CGGGTGCACCGGTCACGCT SEQ ID NO: 241 CTGGGTGGCGGTGGA 3′ GATGTCTCCACCGCC 3′ SEQ ID NO: 271 SEQ ID NO: 272 CETP TP20 Epitope 5′GGCCGGCGGTGGAGACA 5′CGCGTCCACCGCCACCG Type I DISVTGAPVITA TCAGCGTGACCGGTGCACC GCGGTGATCACGGGTGCAC Ala/Gly SEQ ID NO: 242 CGTGATCACCGCCGGTGGC CGGTCACGCTGATGTCTCC GGTGGA 3′ ACCGCC 3′ SEQ ID NO: 273 SEQ ID NO: 274 Ritsch-1 Epitope 5′GGCCGGCGGTGGAGACC 5′CGCGTCCACCGCCACCG Type I DQSVDFEIDSA AGAGCGTGGACTTCGAGAT GCGCTGTCGATCTCGAAGT Ala/Gly SEQ ID NO: 243 CGACAGCGCCGGTGGCGGT CCACGCTCTGGTCTCCACC GGA 3′ GCC 3′ SEQ ID NO: 275 SEQ ID NO: 276

The following sequences, which are human homologues to the corresponding rabbit CETP sequences can be integrated into the AAV2 capsid at position I-453 according to the methods described above:

TABLE 16 CETP derived epitopes at position I-453 Epitope Rabbit Sequence Human Sequence CETP CDAGSVRTNAPD CDSGRVRTDAPD intern SEQ ID NO: 123 SEQ ID NO: 223 CETP FPKHLLVDFLQSLS FPEHLLVDFLQSLS C-Term SEQ ID NO: 261 SEQ ID NO: 224 TP10 AKAVSNLTESRSESLQS PKTVSNLTESSSESVQS SEQ ID NO: 237 SEQ ID NO: 214 TP11 SLTGDEFKKVLET SLMGDEFKAVLET SEQ ID NO: 238 SEQ ID NO: 215 TP12 REAVAYRFEED QHSVAYTFEED SEQ ID NO: 239 SEQ ID NO: 216 TP13 INPEIITLDG INPEIITRDG SEQ ID NO: 240 SEQ ID NO: 217 TP18 DISVTGAPVITATYL DISLTGDPVITASYL SEQ ID NO: 241 SEQ ID NO: 218 TP20 DISVTGAPVITA DISLTGDPVITA SEQ ID NO: 242 SEQ ID NO: 219 Ritsch-1 DQSVDFEIDSA DQSIDFEIDSA SEQ ID NO: 243 SEQ ID NO: 220 Ritsch-2 KNVSEAFPLRAFPPGLLGDS KNVSEDLPLPTFSPTLLGDS SEQ ID NO: 262 SEQ ID NO: 221 Ritsch-3 KNVSEAFPLRA KNVSEDLPLPT SEQ ID NO: 244 SEQ ID NO: 222 Insertion of CETP Epitopes into the AAV2 Capsid at Position I-453 and I-587

Using the cloning strategy described in 9, the following AAV2 capsid variants carrying rabbit CETP epitopes at position I-453 and I-587 were produced:

TABLE 17 CETP double insertion mutants Name Epitope at I-453 Epitope at I-587 AAV-TP10-2x AKAVSNLTESRSESLQS AKAVSNLTESRSESLQS SEQ ID NO: 237 SEQ ID NO: 237 AAV-TP11-2x SLTGDEFKKVLET SLTGDEFKKVLET SEQ ID NO: 238 SEQ ID NO: 238 AAV-TP12/13 REAVAYRFEED INPEIITLDG SEQ ID NO: 239 SEQ ID NO: 240 AAV-TP12-2x REAVAYRFEED REAVAYRFEED SEQ ID NO: 239 SEQ ID NO: 239 AAV-TP13-2x INPEIITLDG INPEIITLDG SEQ ID NO: 240 SEQ ID NO: 240 AAV-TP18-2x DISVTGAPVITATYL DISVTGAPVITATYL SEQ ID NO: 241 SEQ ID NO: 241 AAV-TP20-2x DISVTGAPVITA DISVTGAPVITA SEQ ID NO: 242 SEQ ID NO: 242 AAV-Ritsch1-2x DQSVDFEIDSA DQSVDFEIDSA SEQ ID NO: 243 SEQ ID NO: 243 AAV2-CETin-2x CDAGSVRTNAPD CDAGSVRTNAPD SEQ ID NO: 123 SEQ ID NO: 123 Insertion of Human IgE Epitopes into the AAV2 Capsid at Position I-587

The following human IgE derived epitopes were cloned into position I-587 of the AAV2 capsid using annealed oligonucleotides as described above and were used for production of AAV particles. Each of the inserted epitope sequences is flanked by one of the following alanine/glycine adaptors according to this section 6.4.4 for I-587 above.

TABLE 18 human IgE derived epitopes in I-587 Name/ sense anti-sense Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor 3DEpi3 Epitope 5′ GGCCGGCGGAGGTGGTG 5′ CGCGCACCGCCACCCCC Type II ACAGCAACCCTAGAGGCGT TCTGCTCAGGTAGGCGCTC GAGCGCCTACCTGAGCAGA ACGCCTCTAGGGTTGCTGT GGGGGTGGCGGTG 3′ CACCACCTCCGCC 3′ SEQ ID NO: 277 SEQ ID NO: 278 Wang-CS Epitope 5′ GGCCGGCGGAGGTACCC 5′ CGCGCACCGCCACCCCC Type I ACCCCCACCTGCCCAGAGC GCTTCTCATCAGGGCTCTG CCTGATGAGAAGCGGGGGT GGCAGGTGGGGGTGGGTAC GGCGGTG 3′ CTCCGCC 3′ SEQ ID NO: 279 SEQ ID NO: 280 Flex Epitope 5′ GGCCGGCGGAGGTGAGG 5′ CGCGCACCGCCACCCCC Type I ACGGCCAGGTGATGGACGT GCTCAGGTCCACGTCCATC GGACCTGAGCGGGGGTGGC ACCTGGCCGTCCTCACCTC GGTG 3′ CGCC3′ SEQ ID NO: 281 SEQ ID NO: 282 Bind2 Epitope 5′ GGCCGGCGGAGGTGAGA 5′ CGCGCACCGCCACCACC Type I AGCAGAGAAACGGCACCCT GGTCAGGGTGCCGTTTCTC GACCGGTGGTGGCGGTG TGCTTCTCACCTCCGCC 3′ 3′ SEQ ID NO: 283 SEQ ID NO: 284 C21 Epitope 5′ GGCCGGCGGAGGTGGTC 5′ CGCGCACCGCCACCGGC Type III TGCCCAGAGCCCTGATGAG GCTTCTCATCAGGGCTCTG AAGCGCCGGTGGCGGTG GGCAGACCACCTCCGCC 3′ 3′ SEQ ID NO: 285 SEQ ID NO: 286 Insertion of Cytokine Epitopes into the AAV2 Capsid at Position I-587

The following murine cytokine derived epitopes were cloned into position I-587 of the AAV2 capsid using annealed oligonucleotides as described above and were used for production of AAV particles. Each of the inserted epitope sequences is flanked by one of the following alanine/glycine adaptors according to this section 6.4.4 for I-587 above.

TABLE 19 murine cytokine derived epitopes in I-587 Name/ sense anti-sense Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor mTNFα-V1 Epitope 5′ GGCCGGCGGAGGTAGCA 5′ CGCGCACCGCCACCCCC Type I SSQNSSDKPV GCCAGAACAGCAGCGACAA CTCCACCTGGTGGTTAGCC AHVVANHQVE GCCCGTGGCCCACGTGGTG ACCACGTGGGCCACGGGCT SEQ ID NO: GCTAACCACCAGGTGGAGG TGTCGCTGCTGTTCTGGCT 287 GGGGTGGCGGTG 3′ GCTACCTCCGCC 3′ SEQ ID NO: 294 SEQ ID NO: 295 mTNFα-V2 Epitope 5′ GGCCGGCGGAGGTAGCC 5′ CGCGCACCGCCACCCCC Type I SQNSSDKPVA AGAACAGCAGCGACAAGCC GTGGTTAGCCACCACGTGG HVVANH CGTGGCCCACGTGGTGGCT GCCACGGGCTTGTCGCTGC SEQ ID NO: AACCACGGGGGTGGCGGTG TGTTCTGGCTACCTCCGCC 288 3′ 3′ SEQ ID NO: 296 SEQ ID NO: 297 mTNFα-V3 Epitope 5′ GGCCGGCGGAGGTAGCA 5′ CGCGCACCGCCACCCCC Type I SSQNSSDKP GCCAGAACAGCAGCGACAA GGGCTTGTCGCTGCTGTTC SEQ ID NO: 289 GCCCGGGGGTGGCGGTG TGGCTGCTACCTCCGCC 3′ 3′ SEQ ID NO: 298 SEQ ID NO: 299 mIL-17-V1 Epitope 5′ GGCCGGCGGAGGTAACG 5′ CGCGCACCGCCACCCCC Type I NAEGKLDHHM CCGAGGGCAAGCTTGACCA CAGCACGCTGTTCATGTGG NSVL CCACATGAACAGCGTGCTG TGGTCAAGCTTGCCCTCGG SEQ ID NO: 290 GGGGGTGGCGGTG 3′ CGTTACCTCCGCC 3′ SEQ ID NO: 300 SEQ ID NO: 301 mIL-17-V2 Epitope 5′ GGCCGGCGGAGGTGAGG 5′ CGCGCACCGCCACCCCC Type I EGKLDHHMNSV GCAAGCTTGACCACCACAT CACGCTGTTCATGTGGTGG SEQ ID NO: 291 GAACAGCGTGGGGGGTGGC TCAAGCTTGCCCTCACCTC GGTG 3′ CGCC 3′ SEQ ID NO: 302 SEQ ID NO: 303 mIL-6-V1 Epitope 5′ GGCCGGCGGAGGTAAGA 5′ CGCGCACCGCCACCCCC Type I KSLEEFLKVTL GCCTGGAGGAATTCCTGAA CTGTCTGGTGCTTCTCAGG RSTRQ GGTGACCCTGAGAAGCACC GTCACCTTCAGGAATTCCT SEQ ID NO: 292 AGACAGGGGGGTGGCGGTG CCAGGCTCTTACCTCCGCC 3′ 3′ SEQ ID NO: 304 SEQ ID NO: 305 mIL-6-V2 Epitope 5′ GGCCGGCGGAGGTCTGG 5′ CGCGCACCGCCACCCCC Type I LEEFLKVTLRS AGGAATTCCTGAAGGTGAC GCTTCTCAGGGTCACCTTC SEQ ID NO: 293 CCTGAGAAGCGGGGGTGGC AGGAATTCCTCCAGACCTC GGTG 3′ CGCC 3′ SEQ ID NO: 306 SEQ ID NO: 307

The following sequences, which are human homologues to the corresponding murine cytokine sequence can be integrated into the AAV2 capsid at position I-587 according to the methods described above:

TABLE 20 cytokine derived epitopes Cytokine murine epitope human epitope TNF-α V1 SSQNSSDKPVAHVVANHQVE SSRTPSDKPVAHVVANPQAE SEQ ID NO: 287 SEQ ID NO: 226 TNF-α V2 SQNSSDKPVAHVVANH SRTPSDKPVAHVVANP SEQ ID NO: 288 SEQ ID NO: 227 TNF-α V3 SSQNSSDKP SSRTPSDKP SEQ ID NO: 289 SEQ ID NO: 228 IL-17 V1 NAEGKLDHHMNSVL NADGNVDYHMNSVP SEQ ID NO: 290 SEQ ID NO: 229 IL-17 V2 EGKLDHHMNSV DGNVDYHMNSV SEQ ID NO: 291 SEQ ID NO: 230 IL-6 V1 KSLEEFLKVTLRSTRQ RSFKEFLQSSLRALRQ SEQ ID NO: 292 SEQ ID NO: 231 IL-6 V2 LEEFLKVTLRS FKEFLQSSLRA SEQ ID NO: 293 SEQ ID NO: 232 Insertion of Cytokine Epitopes into the AAV2 Capsid at Position I-453

The following murine cytokine derived epitopes were cloned into position I-453 of the AAV2 capsid using annealed oligonucleotides as described above. Each of the inserted epitope sequences in the AAV2 backbone at I-453 is flanked by the alanine/glycine adaptors according this section 6.4.4 for I-453 above.

TABLE 21 murine cytokine derived epitopes in I-453 Name/ sense anti-sense Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor mTNFα-V1 Epitope 5′ GGCCGCCGGTGGAGGCA 5′ CGCGCCCTCCACCGCCC Type II SSQNSSDKPVA GCAGCCAGAACAGCAGCGA TCCACCTGGTGGTTAGCCA Ala/Gly HVVANHQVE CAAGCCCGTGGCCCACGTG CCACGTGGGCCACGGGCTT SEQ ID NO: 287 GTGGCTAACCACCAGGTGG GTCGCTGCTGTTCTGGCTG AGGGCGGTGGAGGG 3′ CTGCCTCCACCGGC 3′ SEQ ID NO: 308 SEQ ID NO: 309 mIL-17-V1 Epitope 5′ GGCCGCCGGTGGAGGCA 5′ CGCGCCCTCCACCGCCC Type II NAEGKLDHHMN ACGCCGAGGGCAAGCTTGA AGCACGCTGTTCATGTGGT Ala/Gly SVL CCACCACATGAACAGCGTG GGTCAAGCTTGCCCTCGGC SEQ ID NO: 290 CTGGGCGGTGGAGGG 3′ GTTGCCTCCACCGGC 3′ SEQ ID NO: 310 SEQ ID NO: 311 mIL-6-V2 Epitope 5′ GGCCGCCGGTGGAGGCC 5′ CGCGCCCTCCACCGCCG Type II LEEFLKVTLRS TGGAGGAATTCCTGAAGGT CTTCTCAGGGTCACCTTCA Ala/Gly SEQ ID NO: 293 GACCCTGAGAAGCGGCGGT GGAATTCCTCCAGGCCTCC GGAGGG 3′ ACCGGC 3′ SEQ ID NO: 312 SEQ ID NO: 313

The following sequences, which are homologues to the corresponding murine cytokine sequences, can be integrated into the AAV2 capsid at position I-453 according to the methods described above:

TABLE 22 human cytokine derived epitopes in I-453 Cytokine murine epitope human epitope TNF-α V1 SSQNSSDKPVAHVVANHQVE SSRTPSDKPVAHVVANPQAE SEQ ID NO: 287 SEQ ID NO: 226 TNF-α V2 SQNSSDKPVAHVVANH SRTPSDKPVAHVVANP SEQ ID NO: 288 SEQ ID NO: 227 TNF-α V3 SSQNSSDKP SSRTPSDKP SEQ ID NO: 289 SEQ ID NO: 228 IL-17 V1 NAEGKLDHHMNSVL NADGNVDYHMNSVP SEQ ID NO: 290 SEQ ID NO: 229 IL-17 V2 EGKLDHHMNSV DGNVDYHMNSV SEQ ID NO: 291 SEQ ID NO: 230 IL-6 V1 KSLEEFLKVTLRSTRQ RSFKEFLQSSLRALRQ SEQ ID NO: 292 SEQ ID NO: 231 IL-6 V2 LEEFLKVTLRS FKEFLQSSLRA SEQ ID NO: 293 SEQ ID NO: 232 Insertion of Cytokine Epitopes into the AAV2 Capsid at Position I-453 and I-587

Using the cloning strategy described in 9, the following AAV variants carrying different cytokine epitopes at position I-453 and I-587 can be generated (bivalent vaccines):

TABLE 23 double insertion variants for cytokine derived epitopes combination Epitope at I-453 Epitope at I-587 TNF-α/IL-17 mTNFα-V1 mIL-17-V1 SSQNSSDKPVAHVVANHQVE NAEGKLDHHMNSVL SEQ ID NO: 287 SEQ ID NO: 290 TNF-α/IL-6 mTNFα-V1 mIL-6-V2 SSQNSSDKPVAHVVANHQVE LEEFLKVTLRS SEQ ID NO: 287 SEQ ID NO: 293 IL-17/TNF-α mIL-17-V1 mTNFα-V1 NAEGKLDHHMNSVL SSQNSSDKPVAHVVANH SEQ ID NO: 290 QVE SEQ ID NO: 287 IL-6/TNF-α mIL-6-V2 mTNFα-V1 LEEFLKVTLRS SSQNSSDKPVAHVVANH SEQ ID NO: 293 QVE SEQ ID NO: 287 IL-17/IL-6 mIL-17-V1 mIL-6-V2 NAEGKLDHHMNSVL LEEFLKVTLRS SEQ ID NO: 290 SEQ ID NO: 293 IL-6/IL-17 mIL-6-V2 mIL-17-V1 LEEFLKVTLRS NAEGKLDHHMNSVL SEQ ID NO: 293 SEQ ID NO: 290

7. Generation of an Chimeric AAV2 Rep/AAV1 Cap Vector

The approach described below is used for the generation of expression plasmids for the production of AAV1 capsids. This strategy includes the generation of a NotI and AscI restriction site within the cap gene by site-directed mutagenesis that allows the insertion of DNA fragments encoding an epi- or mimotope C-terminally of amino acids S₅₈₈ or D₅₉₀ of AAV1 Cap flanked by a glycine adaptor sequence.

7.1. Substitution of AAV2 Cap by AAV1 Cap within pUC“rep/fs/cap”

Cloning of vector pUCrep/fs/cap is described in detail in US 2004/0087026 (section 0124 and previous sections, there referred to as pUC“rep/fs/cap” Δ37). The complete AAV1 cap ORF, as published by Xiao et al. (Xiao et al., 1999), was amplified by PCR using Expand High FidelityPlus PCR System (Roche; #03300242001). Using specificly modified primers restriction sites were inserted into the cap fragment. SwaI was inserted N-terminally from the VP-1 ATG and NdeI was inserted C-terminally from the polyA site using the primers:

AAV1 Swa for: (SEQ ID NO: 140) 5′-GAT TTA AAT CAG GTA TGG CGT CCG ATG-3′ AAV1 Nde back: (SEQ ID NO: 141) 5′-ACC GAT AA C ATA TGA AGG ACA GGA G-3′

The original sequence of AAV1's N-terminus (Seq. GP-No. 9632548) therefore was modified to read:

(SEQ ID NO: 142)

The original sequence AAV1's C-terminus therefore was modified to read:

(SEQ ID NO: 143)

The PCR fragment was purified and digested with the restriction enzymes SwaI and NdeI (New England Biolabs) according to the instructions of the manufacturer. The same digestion was performed with pUC“rep/fs/cap”. Since SwaI is not a single cutting enzyme in pUC“rep/fs/cap” a partial digestion of NdeI-linearized pUC“rep/fs/cap” was performed with SwaI. The PCR fragment and the desired backbone fragment pUC“rep/fs/cap” of 5077 by (SwaI cut in pUCrep/fs/cap at by 7311) were excised and purified using a Qiagen Gelextraction Kit (Qiagen #28104). PCR fragment and backbone were ligated using the Rapid DNA Ligation Kit (Roche #11 635 379 001) according to manufacturer's protocol. The resulting vector is referred to as pUCrep/fs/cap_AAV1.

7.2. Substitution of AAV2 Cap by AAV1 Cap within pUCAV_AgeI

Cloning of vector pUCAV2_AgeI is described in detail in 6.3. The complete AAV1 cap ORF, as published by Xiao et al. (Xiao et al., 1999), was amplified by PCR using standard procedures using Expand High FidelityPlus PCR System (Roche; #03300242001). Using specifically modified primers restriction sites were inserted into the cap fragment. SwaI was inserted N-terminally from the VP-1 ATG and SnaBI was inserted C-terminally from the polyA site using the primers:

AAV1 Swa for: (SEQ ID NO: 140) 5′-GAT TTA AAT  CAG GTA TGG CGT CCG ATG-3′ AAV1 SnaBI back: (SEQ ID NO: 144) 5′-CGA TAA GAT ACG TAG GAC AGG AGA C-3′

The original sequence of AAV1's N-terminus was therefore modified to read as described in 7.1.

The original sequence AAV1's C-terminus therefore was modified to read:

(SEQ ID NO: 145)

The PCR fragment was purified and digested with the restriction enzymes SwaI and SnaBI (New England Biolabs) according to the instructions of the manufacturer. The same digestion was performed with pUAV2_AgeI. Complete digests were analyzed in an agarose gel, and PCR fragment and the desired backbone fragment of pUCAV2_AgeI were purified utilizing a Qiagen Gelextraction Kit (Qiagen #28104). PCR fragment and backbone were ligated using the Rapid DNA Ligation Kit (Roche #11 635 379 001) according to manufactures protocol. The resulting vector is referred to as pUAV1_AgeI.

7.3. Creation of Singular NotI and AscI Restriction Sites at Amino Acid Position S₅₅₈ or D₅₉₀ within AAV1 Cap

To introduce NotI and AscI restriction sites that allow the cloning of epitope or mimotope sequences C-terminally of amino acid S₅₈₈ or D₅₉₀ of the AAV1 capsid, the vector pUCrep/fs/cap_AAV1 was modified by site directed mutagenesis using the primers:

AAV1 590 NotI AscI for: (SEQ ID NO: 146) 5′-ttc cag agc agc agc aca gac gcg gcc gca aag gcg cgc cct gcg acc gga gat gtg cat-3′ AAV1 590 NotI AscI reverse: (SEQ ID NO: 147) 5′-atg cac atc tcc ggt cgc agg gcg cgc ctt tgc ggc cgc gtc tgt gct gct gct ctg gaa-3′ AAV1 588 NotI AscI for: (SEQ ID NO: 148) 5′-gtc aat ttc cag agc agc agc gcg gcc gca agg cgc gcc aca gac cct gcg acc gga gat-3′ AAV1 588 NotI AscI reverse: (SEQ ID NO: 149) 5′-atc tcc ggt cgc agg gtc tgt ggc gcg cct tgc ggc cgc gct gct gct ctg gaa att gac-3′

Underlined are the sequences of the inserted NotI or AscI restriction sites.

Site directed mutagenesis was performed using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. The resulting vectors are referred to as pUCrep/fs/cap_AAV1_I588 and pUCrep/fs/cap_AAV1_(—)1590, respectively.

7.4. Cloning of AAV1 Variants

7.4.1. Cloning of Rabbit CETP-Intern Epitope Sequence into pUCrep/fs/cap_AAV1_I588 or pUCrep/fs/cap_AAV1_I590

For cloning of the rabbit CETP-intern sequence (CDAGSVRTNAPD, SEQ ID NO: 123) into pUCrep/fs/cap_AAV1_I588 and pUCrep/fs/cap_AAV1_I590, respectively, forward and reverse oligonucleotides were designed that encode the respective CETPint sequence with an adaptor sequence of three glycin residues at each side and containing a 5′-site extension. The 5′-site extension of the oligonucleotides was designed so that annealing of the forward and reverse oligonucleotides results in a dsDNA with 5′-site and 3′-site overhangs compatible with overhangs generated by NotI and AscI restriction of the plasmid pUCrep/fs/cap_AAV1_I588 and pUCrep/fs/cap_AAV1_I590, respectively.

Oligonucleotides

CE_int_I590 AAV1 for: (SEQ ID NO: 150) 5′-G gcc gca ggc ggt gga tgc gac gct ggc agt gtg cgc acc aat gca cca gac  ggc ggt gga gcgg-3′ CE_int_I590 AAV1 rev: (SEQ ID NO: 151) 5′-Cg cgc cgc tcc acc gcc gtc tgg tgc att ggt gcg cac act gcc agc gtc gca  tcc acc gcc tgc-3′ CE_int_I588 AAV1 for: (SEQ ID NO: 152) 5′-G gcc gca ggc ggt gga tgc gac gct ggc agt gtg cgc acc aat gca cca gac  ggc ggt gga gcg-3′ CE_int_I588 AAV1 rev: (SEQ ID NO: 153) 5′-C gcg cgc tcc acc gcc gtc tgg tgc att ggt gcg cac act gcc agc gtc gca  tcc acc gcc tgc-3′

Underlined are G-linkers, bold is the inserted CETP sequence.

For protocol for cloning the oligonucleotides into the vector see example 6.4.3 part ‘Cloning of CETP epitope . . . ’.

7.4.2. Cloning of IgE Epitopes (Kricek and 3DEpi3) into pUCrep/fs/cap_AAV1_I588

The strategy for cloning the Kricek sequence VNLTWSRASG (SEQ ID NO: 85) and the 3DEpi3 sequence into pUCrep/fs/cap_AAV1_I-588, respectively, was the same as described for the CETP insertion in 7.4. Regarding the adaptor sequence five glycin residues were incorporated up and downstream from the 3DEpi3 insertion. An alanin linker was designed for the Kricek insertion resulting in 5 alanins up and downstream of the Kricek insertion within the AAV1 sequence. Since the general design for the insertion of oligonucleotides for the 453 insertion in AAV2 is compatible with the 588 insertion in AAV1, oligonucleotides generated originally for AAV2 insertion could be used for AAV1 588 insertion.

Oligonucleotides:

Kricek long AAV1 for (SEQ ID NO: 314) 5′-G GCC GCA GCC GCA GTG AAC CTG ACC TGG AGC AGA GCC TCC GGC GCG GCA GCT GCA GCT-3′ Kricek long AAV1 rev (SEQ ID NO: 315) 5′-C GCG AGC TGC AGC TGC CGC GCC GGA GGC TCT GCT CCA GGT CAG GTT CAC TGC GGC TGC-3′

Underlined are A-linkers, bold is the inserted Kricek sequence.

3DEpi3 453 uni: (SEQ ID NO: 316) 5′-GGCC GGC GGT GGA GGC GGT GAC AGC AAC CCT AGA GGC GTG AGC GCC TAC CTG AGC AGA  GGA GGC GGT GGA GGG-3′ 3DEpi3 453 rev: (SEQ ID NO: 317) 5′-CGCG CCC TCC ACC GCC TCC TCT GCT CAG GTA GGC GCT CAC GCC TCT AGG GTT GCT GTC  ACC GCC TCC ACC GCC-3′

Underlined are G-linkers, bold is the inserted 3DEpi3 sequence.

For protocol for cloning the oligonucleotides into the vector see example 6.4.3 part ‘Cloning of CETP epitope . . . ’.

7.4.3. Subcloning of Rabbit CETP-Intern Epitope from pUCrep/fs/cap_AAV1_I588 or pUCrep/fs/cap_AAV1_I590 into pUCAV1_AgeI

pUCAV1-AgeI was linearized with BsiWI and AgeI and ligated with the BsiWI/AgeI fragment of pUCrep/fs/cap_AAV1_I588 or pUCrep/fs/cap_AAV1_I590 encoding the VP-2 fragment containing the rabbit CETP-intern epitope after the respective amino acid S₅₈₈ or D₅₉₀ according to standard procedures.

7.4.4. Subcloning of Kricek and 3DEpi3 from pUCrep/fs/cap_AAV1_I588 into pUCAV1_AgeI

pUCAV1-AgeI was linearized with BsiWI and AgeI and ligated with the BsiWI/AgeI fragment of pUCrep/fs/cap_AAV1_I588 encoding the VP-2 fragment containing the Kricek and the 3DEpi3 epitope, respectively, according to standard procedures.

7.4.5. Subcloning of NotI and AscI Restriction Sites at Amino Acid Position S₅₈₈ from pUCrep/fs/cap_AAV1_I588 into pUCAV1_AgeI

To allow direct cloning of polypeptides into pUCAV1_AgeI the NotI/AscI insertion site was cloned into pUCAV1_AgeI. Therefore, pUCAV1-AgeI was linearized with BsiWI and AgeI and ligated with the BsiWI/AgeI fragment of pUCrep/fs/cap_AAV1_I588 encoding the VP-2 fragment containing NotI/AscI insertion respectively according to standard procedures.

The resulting vector is named pUCAV1-AgeI-I588.

7.4.6. Cloning of Rabbit CETP Sequence TP18 into pUCAV1-AgeI-I588

The strategy for cloning the TP18 sequence DISVTGAPVITATYL (SEQ ID NO: 241) into pUCAV1-AgeI-I588 respectively was the same as described for the CETP insertion in 7.4. Regarding the adaptor sequence three glycin residues were incorporated up and 4 glycin residues downstream from the TP18 insertion. Since the general design for the insertion of oligonucleotides for the 453 insertion in AAV2 is compatible with the 588 insertion in AAV1, oligonucleotides generated originally for AAV2 insertion could be used for AAV1 588 insertion.

Oligonucleotides:

TP18-453 uni (SEQ ID NO: 318) 5′-GGCC GGC GGT GGA GAC ATC AGC GTG ACC GGT GCA CCC GTG ATC ACC GCC ACC TAC CTG  GGT GGC GGT GGA- 3′ TP18-453 rev (SEQ ID NO: 319) 5′-CGCG TCC ACC GCC ACC CAG GTA GGT GGC GGT GAT CAC GGG TGC ACC GGT CAC GCT GAT GTC  TCC ACC GCC- 3′

Underlined are G-linkers, bold is the inserted TP18 sequence.

For protocol for cloning the oligonucleotides into the vector see example 6.4.3 part ‘Cloning of CETP epitope . . . ’.

7.5. Production of AAV1 Variants by Co-Transfection of HEK 293-T-Cells

For production of AAV particles HEK 293-T cells were co-transfected with the vector plasmid pUCAV1 or pUCrep/fs/cap_AAV1 with or without the subcloned epitope (after amino acids S₅₈₈ or D₅₉₀) and the helper plasmid pUCAdV. For the production of pUCrep/fs/cap_AAV1 derived capsids (with or without the subcloned epitope) pGFP was additionally transfected since pUCrep/fs/cap_AAV1 does not encode for the AAV ITRs which function as a packaging signal. pGFP encodes GFP flanked by the AAV2 ITRs. Thus GFP is packaged as a transgene.

Resulting viral particles carrying the rabbit CETP epitope CETP-intern were named: AAV1-CETP-588 and AAV1-CETP-590 which were derived from pucAV1 derivates and rAAV1-GFP-CETP-588, rAAV1-GFP-CETP-590 which were derived from pUCrep/fs/cap_AAV1 derivates.

Production and purification of viral particles were performed as described above (see example 4).

Surprisingly yields for AAV1-particles were about 3-6 times higher compared to AAV2-particles, making AAV1 mutated structural proteins an especially preferred embodiment of this invention in all its aspects.

7.6. Evaluation of AAV1 Particles Carrying the Rabbit CETP-Intern Epitope After Amino Acids S₅₈₈ or D₅₉₀

The AAV1 capsid variants carrying the CETP-intern epitope at position 588 or 590 were analyzed by dot blot analysis (FIG. 11). 5.0×10¹¹ to 3.1×10¹⁰ AAV capsids were spotted onto a nitrocellulose membrane using a vacuum device. As controls AAV2-CETPin 2× (AAV2 particles carrying the CETP-intern epitope at position I-453 and I-587) and wtAAV1 particles were spotted. After blocking of the membrane with blocking buffer (5% milk powder in PBS containing 0.05% Tween-20) for 1 h at room temperature, the membrane was incubated with the anti-CETP polyclonal rabbit serum (1:2,500 in 1% milk powder in PBS containing 0.05% Tween-20) for 1 h at room temperature. The serum was derived from rabbits vaccinated with the CETP-intern peptide coupled to LPH. After washing the membrane with PBS/0.05% Tween-20, binding of the anti-CETP serum to the spotted AAV variants was detected with an anti-mouse IgG HRP conjugate. The membrane was incubated with an anti-rabbit IgG HRP conjugate (1:2,500 in 1% milk powder in PBS containing 0.05% Tween-20) for 1 h at room temperature. After washing, signals were detected by chemiluminescence using the ECL system (Pierce) (FIG. 11B).

To demonstrate that equal amounts of AAV variants were spotted on the membrane, and to exclude cross reactions of the antibodies, an additional membrane was spotted as described above and spotted AAV capsids were detected using an anti-AAV1 antibody recognizing intact AAV1 particle (Progen) (FIG. 11B). For this, the membrane was incubated with the anti-antibody (Progen) (1:500 diluted in 1% milk powder in PBS containing 0.05% Tween-20) for 1 h at room temperature. After washing of the membrane with PBS/0.05% Tween-20, binding of the AAV1 mAb to the spotted AAV variants was detected with an anti-mouse IgG (γ) HRP conjugate (CALTAG). The membrane was incubated with the anti-mouse IgG (γ) HRP conjugate for 1 h at room temperature. After washing, signals were detected by chemiluminescence using the ECL system (Pierce) (FIG. 11A).

The results demonstrate that AAV1 CETP variants are specifically detected by the anti-CETP serum indicating that the CETP epitope inserted at both positions (after amino acids S₅₅₅ and D₅₉₀) is displayed on the surface of the capsid.

7.7. Analysis of Cross-Reactivity of AAV1 Capsids with Serum of AAV2 Vaccinated Rabbits

Wild-type AAV2 and AAV1 capsids were coated onto Maxisorp 96 well plates (Nunc). Capsids were coated in equal amounts in serial dilutions from 1.0×10⁹ to 1.56×10⁷ capsids per well for 1 h at 37° C. After blocking with blocking buffer (5% milk powder in PBS containing 0.05% Tween-20) for 1 h at 37° C., wells were incubated with sera from rabbits vaccinated with AAV2 (1:400 in 1% milk powder in PBS containing 0.05% Tween-20) for 1 h at 37° C. After washing the wells with PBS/0.05% Tween-20, binding of the polyclonal rabbit serum to the coated AAV variants were detected with an anti-rabbit IgG HRP conjugate (DAKO). Wells were incubated with the anti-rabbit IgG HRP conjugate for 1 h at room temperature. After washing, substrate (TMB) was added to the wells. The reaction was stopped after 15 min by adding 0.2 M H₂SO₄. OD at 450 nm was measured in an ELISA reader.

The result demonstrates that serum from AAV2 vaccinated rabbits binds less efficiently to AAV1 (up to factor 8 regarding the OD values) compared to AAV2 (FIG. 12) confirming that AAV1 and AAV2 are different serotypes with little cross-reactivity of anti-capsid antibodies. Therefore, it can be concluded that the cross-reactive antibodies in AAV2 sero-positive humans have only limited neutralizing activity on AAV1 vaccines applied to humans. As (neutralizing) antibodies against the viral backbone can limit both vaccination and gene transfer efficacy, this indicates that AAV1 is a preferable serotype for treatment of AAV2 sero-positive humans, both regarding vaccination and gene transfer.

These results were confirmed in a similar experiment, where same amounts of capsids (1×10⁹) of rAAV2-GFP, rAAV1-GFP, rAAV1-GFP-CETP-588 and rAAV1-GFP-CETP-590 were coated onto Maxisorp 96 well plates (Nunc) and incubated with serial dilutions of sera from rabbits vaccinated with AAV2 (1:100-1:6400). The binding assay was performed as described above (FIG. 13).

This experiment further confirms the results above and shows additionally that the CETP insertion does not interfere with this result.

8. Tools to Study Anti-IqE Antibodies 8.1. Generation of 293 Cells Overexpressing the α- and γ-Chain of Human FcεRI

The cDNA of the α-chain of human FεεRI (FcεRIα) (including the stop-codon) cloned into pENTR™ 221 was obtained from Invitrogen and was sub-cloned into the expression vector pEF5/FRT/V5-Dest (Invitrogen) using the Gateway Cloning System (Invitrogen) according to the instructions of the manufacturer. The resulting expression vector is referred to as pEF5-FcεRIα. The FcεRIα cDNA is expressed under the control of the eukaryotic EF1α promoter in this vector.

Flp-In™ 293 cells (Invitrogen) were transfected with the vector pEF5-FcεRIα using Lipofectamine™ 2000. 4×10⁵ cells were seeded into one well of a 6-well cell culture plate in a total volume of 2.0 ml DMEM supplemented with 10% FCS, 5 mM glutamine, NEAA (1×) (non-essential amino acids) and 100 μg/ml zeocin. After 24 h of cultivation, medium was replaced with serum-free DMEM and cells were transfected with a mixture of 10 μl lipofectamine, 2 μg vector pEF5-FcεRIα and 2 μg vector pOG44 (Invitrogen) in a total volume of 100 μl MEM according to the instructions of the manufacturer.

The vector pOG44 encodes a recombinase (Flp recombinase) that mediates the integration of a vector containing the gene of interest (FcεRIα) and a FRT site into the genome of the Flp-In™ 293 cells via Flp Recombination Target (FRT) sites. After 6 h FCS was added to the cells to a final concentration of 5%. 48 h after transfection cells were split in a 1:10 ratio and cultivated in DMEM, 10% FCS, 5 mM glutamine, NEAA (1×) and 100 μg/ml hygromycin B to select transfected cells. Single stably transfected cell clones were isolated by sub-culturing of picked cell clusters in DMEM, 10% FCS, 5 mM glutamine, NEAA (1×) and 100 μg/ml hygromycin B.

Integration of the FcεRIα cDNA into the genome of the cells was analyzed by PCR. Genomic DNA of the transfected cells was isolated using the DNeasy Tissue DNA Isolation Kit (Qiagen). PCR was performed using the primers

FcεRlα-uni (SEQ ID NO: 154) 5′-TGT GTG TAG CCT TAC TGT TCT TCG C-3′ and FcεRlα-rev (SEQ ID NO: 155) 5′-CTTCTCACGCGGAGCTTTTATTAC-3′ and a Taq Polymerase Mastermix (Qiagen). Since the primers are located at exon-intron boundaries of the human FcεRIα gene, only the cDNA of FcεRIα integrated into the genome of the cells is amplified by PCR.

Although the cDNA of FεRIα was stably integrated into the genome of the transfected cells, no significant cell surface expression of FcεRIα could be detected by flow cytometry using a PE-labeled FcεRIα specific mAb (eBioscience) at a final concentration of 2.5 μg/ml in PBS supplemented with 0.5% BSA.

Since co-expression of the γ-chain of FcεRI is known to increase cell surface expression of FcεRIα (Kuster et al., 1990), a single 293 cell clone stably transfected with the α-chain (clone A3) was transfected with the cDNA of FcεRIγ. The cDNA of FcεRIγ (including stop-codon) cloned into the vector pENTR™ 221 was obtained from Invitrogen and was sub-cloned into the expression vectors pEF-DEST51 (Invitrogen) and pcDNA6.2-V5-DEST (Invitrogen). The cDNA is expressed under the control of the eukaryotic EF1α promoter or the CMV promoter in pEF-DEST51 or pcDNA6.2-V5-DEST, respectively. The 293 cell clone A3 was transfected with the vectors pEF-FcεRIγ or pcDNA6.2-FcεRIγ, respectively, using Lipofectamine™ 2000 and 4 μg of the vector as described above. Transfected cells expressing the α- and γ-chain of FcεRI were selected by cultivation of the cells in DMEM with 10% FCS, 5 mM glutamine, NEAA (1×), 100 μg/ml hygromycin B and 5 μg/ml blasticidin (selection medium). Single stably transfected cell clones were isolated by sub-culturing of picked cell clusters of the transfected cell pool in the selection medium.

FcεRIα cell surface expression of the cell clones was monitored by flow-cytometry using a PE-labeled anti-human FcεRIα mAb (eBioscience) at a final concentration of 2.5 μg/ml in PBS supplemented with 0.1% BSA (FIG. 15). IgE binding of the cells was analyzed by incubation of 1.0×10⁵-5.0×10⁵ cells with biotin-labeled human IgE (Dianova) at a concentration of 20 μg/ml in a total volume of 100 μl PBS, 0.5% BSA (incubation buffer) for 30 min at room temperature (RT). After washing of the cells with incubation buffer, IgE binding was detected by flow-cytometry using PE-labeled streptavidin (CALTAG) at 15 μg/ml (in 20 μl incubation buffer). Cells were stained with the PE-labeled streptavidin for 30 min on ice (data not shown).

The results demonstrate that co-expression of the γ-chain increases the cell surface expression of the FcεRIα-chain. The increased cell surface expression is associated with an increased binding of human IgE by the transfected cells demonstrating that the cell surface exposed α-chain is functionally active. The individual cell clones differed with respect to the cell surface expression of FcεRIα and the clone showing the highest expression and IgE binding was selected for subsequent assays.

To evaluate the effect of anti-IgE antibodies on binding of human IgE to FcεRIα, the cell clone D11 co-expressing the α-chain (under control of EF1a promoter) and the γ-chain (under control of a CMV promoter) was used for IgE binding assays (FIG. 16). Increasing concentrations of biotin-labeled human IgE (78 ng/ml to 10 μg/ml) were pre-incubated with a constant concentration (2.5 μg/ml) of anti-IgE antibodies Bsw17 (kindly provided by Prof. Stadler, Bern) or XOLAIR® in a total volume of 100 μl incubation buffer for 1.5 h at RT. As a negative control IgE was pre-incubated with mouse IgG₁ under the same conditions. A T175 cell culture flask with 80-90% confluent cells (clone D11) was harvested using Cell Dissociation Buffer (Gibco) and cells were resuspended in 15 ml DMEM medium. 200 μl of this cell suspension was added to each well of a 96-well tissue culture plate. The plate was centrifuged and the cells were washed with incubation buffer and resuspended in 100 μl of the IgE/anti-IgE mixture. Cells were incubated for 30 min a RT. After washing of the cells with incubation buffer, cells were stained with the PE-labeled streptavidin (15 μg/ml) for 30 min on ice. IgE binding was detected by flow-cytometry (FIG. 16).

These data demonstrate that the transfected 293 cells expressing the α- and γ-chain of human FcεRI provide a tool to monitor the binding of human IgE to FcεRIα and the effect of anti-IgE antibodies thereon.

8.2. Generation of RBL2H3 Cells Overexpressing the α-of Human FcεRI

The α-chain of human FcεRI (FcεRIα) (including the stop-codon) cloned into pENTR™ 221 was obtained from Invitrogen and was sub-cloned into the expression vectors pEF-DEST51 (Invitrogen) and pcDNA6.2-V5-DEST (Invitrogen) using the Gateway Cloning System (Invitrogen). Rat basophile RBL2H3 cells (80-90% confluent) were transfected with the resulting vectors pEF-FcεRIα or pcDNA6.2-FcεRIα, respectively, using Lipofectamine™ 2000 and 4 μg of the vector as described above. Transfected cells expressing the α-chain of FcεRI were selected by cultivation of the cells in RPMI with 10% FCS, 5 mM glutamine, 1×NEAA supplemented with 15 μg/ml blasticidin (selection medium). Single stably transfected cell clones were isolated by sub-culturing of picked cell clusters of the transfected cell pool in the selection medium. FcεRIα cell surface expression of the cell clones was monitored by flow-cytometry using a PE-labeled anti-human FcεRIα mAb (eBioscience) at a final concentration of 2.5 μg/ml in PBS supplemented with 0.5% BSA (data not shown).

The cell clone E5 (stably expressing the α-chain under control of an EF1α promoter) was used for evaluation of human IgE-mediated histamine release. 1.0×10⁴ cells were seeded into a well of 96-well tissue culture plate and cultivated in a total volume of 200 μl RPMI/10% FCS/5 mM glutamine/1× NEAA in a humidified atmosphere at 37° C. and 5.0% CO₂. Cells were sensitized by cultivation in the presence of human IgE (Dianova) at increasing concentrations (0.08-10.0 μg/ml) in complete RPMI medium for 2 h or 48 h in a total volume of 250 μl. Cells were washed with Tyrode's Salt Solution (Sigma) supplemented with 0.1% BSA and histamine release was induced by cross-linking of receptor-bound human IgE by the anti-human IgE antibody Le27 (100 nM) (kindly provided by Prof. Stadler, Bern; (Grassi et al., 1986)) in a total volume of 100 μl Tyrode's Salt Solution/0.1% BSA for 1 h. Histamine content of the medium was measured using a commercially available histamine ELISA (Neogen) (FIG. 17).

To evaluate the effect of anti-IgE antibodies on the human IgE-dependent histamine release of the stably transfected RBL2H3 cells, cells (clone E5) were sensitized with 2.0 μg/ml human IgE, which was pre-incubated with XOLAIR® mAb (5.0-25.0 μg/ml) for 2 h at room temperature. For sensitization, cells were cultivated with the IgE/XOLAIR® mAb mixture for 2 h as described above in a total volume of 100 μl RPMI medium. Histamine release was induced by the anti-IgE mAb Le27 as described above and the histamine content of the medium was measured by ELISA (Neogen) (FIG. 18).

These data demonstrate that the transfected RBL2H3 cells expressing the α-chain of human FcεRI can be sensitized with human IgE and can be induced to release histamine in the presence of a human IgE cross-linking agent. The cells provide a tool to study the human IgE-induced degranulation of basophiles and the effect of anti-IgE antibodies thereon.

8.3. In Vitro Binding Assays Using Recombinant FcεRIα

The α-chain of human FcεRI can be expressed as a recombinant protein in prokaryotic or eukaryotic cells. After purification the recombinant FcεRIα can be immobilized on a suitable matrix (e.g. plastic plate, beads). Purification and immobilization can also be performed using a suitable tag fused to the recombinant FcεRIα at the N- or C-terminus (e.g. His-tag, FLAG-tag, S-Tag, GST-tag). The immobilized FcεRIα will be incubated with labeled human IgE. The label can be a, for example, fluorescent dye, biotin, peroxidase or alkaline phosphatase. Binding of IgE will be detected using this label and the appropriate detection system (fluorescence measurement, labeled streptavidin, peroxidase substrate, alkaline phosphatase substrate). To evaluate the effect of anti-IgE antibodies on the interaction of IgE with recombinant FcεRIα, IgE will be preincubated with the anti-IgE antibodies and subsequently used in the binding assay described above.

9. Double Insertion of a β-Amyloid Epitope at Position I-453 and I-587 of the AAV Capsid

The cloning approach described below is used for the double insertion of an epi- or mimotope sequence into the AAV capsid at position I-453 and I-587 using a defined cloning strategy

9.1. Insertion of an FseI Restriction Site into pCIVP2

An FseI restriction site was inserted into the vectors pCIVP2-I587-NotI-AscI and pCIVP2-I453-NotI-AscI located between I-453 and I-587 by site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene) and the oligonucleotides

mutashe-9 (SEQ ID NO: 156) 5′-GGT GAA TCC GGG GCC GGC CAT GGC AAG C-3′ and mutashe-10 (SEQ ID NO: 157) 5′-GCT TGC CAT GGC CGG CCC CGG ATT CAC C-3′. 9.2. Cloning of a β-Amyloid Epitope at Position I-587 of pUCAV2

The β-amyloid epitope DAEFRHDSG (SEQ ID NO: 158) (aa 1-9 of human β-amyloid) was cloned into the NotI/AscI restriction site of the vector pCIVP2-I587-NotI-AscI (modified as described in 9.1) using the sense and anti-sense oligonucleotides

β-amyloid-for (SEQ ID NO: 159) 5′-GGC CGC AGG CGG AGG GGG AGG CGA CGC CGA GTT CAG ACA CGA CAG CGG CGG CGG AGG GGG AGG CGC GG-3′ and β-amyloid-rev (SEQ ID NO: 160) 5′-CGC GCC GCG CCT CCC CCT CCG CCG CCG CTG TCG TGT CTG AAC TCG GCG TCG CCT CCC CCT CCG CCT GC-3′

The oligonucleotides encode the β-amyloid epitope with a glycine adaptor sequence:

(A)₃-(G)₅-DAEFRHDSG-(G)₅-(A)₂ (SEQ ID NO: 161)

Cloning was performed as described above (6.2).

The BsiWI/XmaI fragment of pCI-VP2-587-NotI-AscI encoding a VP-2 fragment containing the β-amyloid epitope at position I-587 was sub-cloned into pUCAV2-AgeI as described above (6.3). The resulting vector was referred to as pUCAV2-amyloid-587.

9.3. Cloning of a β-Amyloid Epitope at Position I-453 of pCIVP2

The β-amyloid epitope (DAEFRHDSG, SEQ ID NO: 158) was cloned into the NotI/AscI restriction site at the insertion site I-453 of the vector pCIVP2-I453-NotI-AscI (modified as described in 9.1) using the sense and anti-sense oligonucleotides

Amyloid 453for (SEQ ID NO: 162) 5′-G GCC GGC GGA GGC GGT GGG GAC GCC GAA TTC AGA CAC GAC AGC GGC GGA GGC GGT GGA GGG-3′ Amyloid 453rev (SEQ ID NO: 163) 5′-C GCG CCC TCC ACC GCC TCC GCC GCT GTC GTG TCT GAA TTC GGC GTC CCC ACC GCC TCC GCC-3′

The oligonucleotides encode the β-amyloid epitope with a glycine adaptor sequence:

(A)₂-(G)₅-DAEFRHDSG-(G)₅-R-(A)₂ (SEQ ID NO: 164)

Cloning was performed as described above (6.2).

9.4. Cloning of a β-Amyloid Epitope at Position I-453 and I-587 of pUCAV2

For production of recombinant AAV particles carrying the β-amyloid epitope at position I-587 and I-453, the vector pUCAV2-amyloid-587 was cut with BsiWI/FseI and ligated with the 0.6 kb BsiWI/FseI fragment of pCI-VP2-453-NotI-AscI. The BsiWI/FseI fragment of pCI-VP2-453-NotI-AscI encodes the VP-2 fragment containing the β-amyloid epitope at position I-453. The resulting vector was referred to as pUCAV2-amyloid-453-587.

9.5. Production, Purification and Evaluation of AAV Particles Carrying a β-Amyloid Epitope at I-453 and I-587

For production of recombinant AAV particles carrying the β-amyloid epitope at position I-587 and I-453, 293 cells were transfected with the vector pUCAV2-amyloid-453-587 and the helper plasmid pUCAdV as described above (4.2 and 4.3). The corresponding AAV particles were referred to as AAV-amyloid-453-587.

For production of recombinant AAV particles carrying the β-amyloid epitope at position I-587, 293 cells were transfected with the vector pUCAV2-amyloid-587 and the helper plasmid pUCAdV as described above. The corresponding AAV particles were referred to as AAV-amyloid-587. All AAV particles were purified as described above

To evaluate the expression of the β-amyloid epitope at the surface of the AAV capsid, serial dilutions of purified AAV particles AAV-amyloid-453-587 and AAV-amyloid-587 were dotted on a membrane (FIG. 19). As a negative control AAV particles carrying a CETP epitope at position I-587 were dotted. As a positive control a β-amyloid peptide (aa I-42) (Biosource) was dotted. After blocking of the membrane with blocking buffer (5% milk powder in PBS containing 0.05% Tween-20), the β-amyloid epitope was detected using an anti-β-amyloid mAb 6E10 (Chemicon) (FIG. 19). The anti-β-amyloid mAb was used at a concentration of 1.0 μg/ml in PBS/1% milk powder/0.05% Tween-20. Binding of the anti-β-amyloid mAb was detected using a peroxidase labeled anti-mouse IgG antibody (CALTAG). After washing, signals were detected by chemiluminescence using the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

These data demonstrate that the double insertion of the epitope into the insertion sites I-453 and I-587 results in higher epitope density at the capsid surface than the singular insertion of the epitope at position I-587.

10. Immunization of Rabbits with AAV-Based Vaccines

10.1. Production and Purification of AAV2-Based Vaccines for Immunization Experiments

For production of AAV particles HEK 293-T cells were co-transfected with the vector plasmid pUCAV2 containing the subcloned epitope (in I-453 and/or I-587) and the helper plasmid pUCAdV as described above. For large scale production 30-60 Ø15 cm cell culture plates with 7.5×10⁶ 293-T cells were seeded and cultivated at 37° C., 5% CO₂ in a humidified atmosphere. Co-transfection of the cells with the vector plasmid pUCAV2 containing the epitope (in I-453 or I-587) and pUCAdV was performed as described above. 72 h after transfection 293-T cells and medium were harvested and centrifuged at 3000 g at 4° C. for 15 min. The cell pellet was resuspended in 15-30 ml lysis buffer (50 mM HEPES, 200 mM NaCl, 2.5 mM MgCl₂; pH 6.8) and objected to three rounds of freeze and thaw cycles. The cleared cell culture supernatant was concentrated by TFF (tangential flow filtration) using the SARTOFLOW® Slice 200 Benchtop Cross-flow system using a SARTOCON® Slice 200 cassette (Hydrosart membrane). The TFF concentrate of the cell culture supernatant (about 35 ml) was pooled with the cleared crude lysate and subsequently treated with 1667 U/ml benzonase (Merck) at 37° C. for 2 h-4 h. After benzonase treatment the pool of crude lysate and TFF concentrate was centrifuged at 3600 g for 5 min at 4° C. The AAV-containing supernatant was separated through a size exclusion chromatography (SEC) column. SEC was performed using a XK50/20 column packed with SUPERDEX 200® resin beads and SEC running buffer (50 mM HEPES, 400 mM NaCl, 2.5 mM MgCl₂; pH 6.8). SEC fractions were analyzed by AAV2 ELISA. AAV-containing fractions were pooled and objected to iodixanol gradient centrifugation. Iodixanol solutions of different concentrations were layered beneath the pool of virus containing SEC fraction in QUICKSEAL® centrifugation tubes (25×89 mm; Beckman). By this an Iodixanol gradient was created composed of 4.0 ml 60% on the bottom, 5.0 ml 40%, 4.0 ml 25% and 5.5 ml 15% Iodixanol with the virus solution on top. The gradient was centrifuged using a fixed angel rotor (Ti 70.1 rotor, Beckman) at 65000 rpm for 1 h at 18° C. The 40% phase containing the AAV particles was then extracted with a cannula by puncturing the tube underneath the 40% phase and allowing the solution to drip into collecting tubes. Fractions of about 0.5 ml were collected until the 25% phase was reached. The AAV capsid titer of the fractions was determined using a commercially available ELISA (AAV Titration ELISA, Progen). Purity of the AAV-containing fractions was determined by SDS-PAGE and subsequent colloidal Coomassie staining. Fractions with high purity of AAV particles were pooled and the capsid titer of the final pool was determined by AAV2 titration ELISA.

10.2. Breaking of Self-Tolerance by AAV-Based Vaccines

A panel of AAV-based vaccines carrying epitopes derived from rabbit CETP was generated as described above. AAV-based CETP vaccines were compared with the corresponding peptide vaccines containing the same epitope coupled to LPH (Limulus polyphemus hemocyanine) as a carrier protein. The peptides were chemically synthesized with a C- or N-terminal Cystein residue that was used for coupling of the peptides to LPH. Synthesis and coupling of the peptides was performed by Biogenes (Berlin, Germany).

The vaccines described in Table 24 were used for immunization of rabbits:

TABLE 24 Vaccines used for immunization of rabbits Name of Vaccine Insertion Dose vaccine carrier Site Epitope (μg) AAV-TP11 AAV2 I-587 SLTGDEFKKVLET 10.9 SEQ ID NO: 238 AAV-TP12 AAV2 I-587 REAVAYRFEED 14.1 SEQ ID NO: 239 AAV-TP13 AAV2 I-587 INPEIITLDG 13.3 SEQ ID NO: 240 AAV-TP18 AAV2 I-587 DISVTGAPVITATYL 7.2 SEQ ID NO: 241 LPH-TP11 LPH N/A CSLTGDEFKKVLET see SEQ ID NO: 320 text LPH-TP12 LPH N/A CREAVAYRFEED see SEQ ID NO: 321 text LPH-TP13 LPH N/A CINPEIITLDG see SEQ ID NO: 322 text LPH-TP18 LPH N/A CDISVTGAPVITATYL see SEQ ID NO: 323 text

For each vaccination approach two rabbits were immunized s.c. with the vaccines shown in the table above four times (one prime and three boost immunizations). The first boost immunization was performed 2 weeks after an initial prime immunization. Rabbits were boosted another two times with the vaccines at intervals of 3 weeks. Serum of the immunized animals was prepared two weeks after each boost immunization.

The purified AAV-based vaccines were mixed an equal volume of formulation buffer (PBS with 1% sorbitol, 0.2% Tween-20, 25% propylenglycol, 200 mM NaCl and 2.5 mM MgCl₂) for stabilization of the particles and stored at −80° C. until administration. If necessary, the volume of the AAV-based vaccines was adjusted to 0.3 ml with formulation buffer directly before application. The vaccines were administered s.c. in the presence of 0.7 ml adjuvant (total volume 1 ml). The adjuvant was provided by Biogenes and contained amongst others 0.01% lipopolysaccharide derived from Phormidium, 95% paraffin oil, 2.4% Tween-40 and 0.1% cholesterol.

The LPH-coupled peptides (in 0.3 ml TBS) were administered s.c. in the presence of 0.7 ml of the adjuvant provided by Biogenes. 1 mg of the LPH-peptide conjugate was administered for the prime immunization. 0.5 mg of the conjugate was used for the 1^(st) boost immunization and 0.25 mg of the conjugate were used for the 2^(nd) and 3^(rd) boost immunization.

Induction of anti-CETP auto-antibodies in the vaccinated animals was determined by ELISA using recombinant rabbit CETP as antigen. For production of rabbit CETP, the CETP cDNA was amplified by RT-PCR using the primers

rCETP-uni (SEQ ID NO: 324) 5′-GGG GAA TTC ATG TCC CAA AGG CGC CTC CTA CG-3′ and rCETP-rev (SEQ ID NO: 325) 5′-GGG GGA TCC CTA GCT CAG GCT CTG GAG GAA ATC C- 3′

and rabbit liver PolyA⁺ RNA (Clontech) as template. The CETP cDNA was cloned into the EcoRI/BamHI site of the vector p3XFLAG-CMV-8 (SIGMA). The resulting vector encodes the mature CETP sequence with a C-terminal FLAG®-tag and an N-terminal preprotrypsin leader sequence for secretion of the recombinant protein. For expression of recombinant rabbit CETP 293T cells were transfected with the vector by calcium phosphate transfection as described above. CETP was purified from the cell culture supernatant by affinity chromatography using anti-FLAG° M2 agarose beads (SIGMA). Purity of the recombinant rabbit CETP was analyzed by SDS-PAGE and subsequent colloidal coomassie staining. CETP activity was determined using a commercially available CETP activity assay (Roar).

For titration of rabbit CETP auto-antibodies in the immune sera, a 96-well Maxisorp plate (Nunc) was coated with purified recombinant rabbit CETP (100 ng/well) for 1 h at 37° C. After coating wells were washed with wash buffer (PBS/0.1% Tween-20) and subsequently incubated with blocking buffer (5% skim milk in wash buffer) for 1 h at 37° C. After blocking of the wells, immobilized CETP was incubated with serial dilutions of the immune sera in dilution buffer (wash buffer with 1% skim milk and 1% BSA) for 1 h at 37° C. Rabbit pre-immune sera or rabbit sera of unrelated vaccinations served as negative controls. After washing binding of rabbit IgG to the immobilized CETP was detected using a HRP-labelled anti-rabbit IgG antibody (H+L) (DAKO; 1:2500 in dilution buffer). Signals (OD) were detected using TMB (KemEnTec) as substrate.

CETP auto-antibody titers were determined by end point dilution. The titer of the immune serum corresponds to the intersection point of the titration curve of the immune sera with the limit of detection of the assay.

The limit of detection (LOD) of the assay was calculated as follows:

Mean OD (unspecific sera)+3.3×standard deviation OD (unspecific sera)

In addition to the CETP auto-antibody titers, the anti-peptide titers of the immune sera were analyzed. The free peptides (corresponding to the epitopes integrated in the AAV capsid or coupled to LPH) were covalently immobilized in a 96-well plate (REACTI-BIND™ Amine-binding, Maleic Anhydride Activated Plates; Pierce). For immobilization of the peptide, the 96-well plate was incubated with 1 μg peptide per well in a total volume of 50 μl PBS for at least 1 h at 37° C. After coating of the peptides wells were blocked with 200 μl/well blocking buffer (PBS/5% skim milk/0.1% Tween-20) for 1 h at 37° C. After blocking of the wells, immobilized peptides were incubated with serial dilutions of the immune sera in dilution buffer (PBS with 1% skim milk, 1% BSA, 0.1% Tween-20) for 1 h at 37° C. Rabbit pre-immune sera or rabbit sera of unrelated vaccinations served as negative controls. After washing binding of rabbit IgG to the immobilized CETP was detected using a HRP-labelled anti-rabbit IgG antibody (DAKO; 1:2500 in dilution buffer). Signals (OD) were detected using TMB (KemEnTec) as substrate. Antibody titers were determined as described above.

Except for one animal vaccinated with AAV-TP13 the data demonstrate that vaccination with AAV-based vaccines induces high titers of target-specific auto-antibodies that are not obtained using peptide-based vaccines. Accordingly, AAV-based vaccines are able to break self-tolerance and induce high levels of auto-antibodies (FIG. 20). The immunogenic properties of the peptide based vaccines are reflected by the high titers of peptide specific antibodies induced by the peptide vaccines (FIG. 21). However, these antibodies show only weak reaction with native rabbit CETP (FIG. 20) suggesting that peptide based vaccines—although immunogenic—have only a limited potential to break self-tolerance and induce low levels of auto-antibodies.

10.3. The AAV Capsid Structure is Essential for Breaking of Self-Tolerance and Induction of Auto-Antibodies

To demonstrate that the capsid structure and the structured, repetitive presentation of epitopes within the AAV-capsid are essential for breaking of self-tolerance of the immune system and induction of auto-antibodies, rabbits were immunized with heat-denatured AAV-TP11-2× or AAV-TP18-2× particles. Results were compared with vaccinations using the corresponding native particles. The AAV-variant AAV-TP11-2× carries the rabbit CETP TP11 epitope (SLTGDEFKKVLET, SEQ ID NO: 238) at positions I-453 and I-587. The AAV-variant AAV-TP18-2× carries the rabbit CETP TP18 epitope (DISVTGAPVITATYL, SEQ ID NO: 241) at positions I-453 and I-587. For heat denaturation the particles were mixed with an equal volume of formulation buffer (PBS with 1% sorbitol, 0.2% Tween-20, 25% propylenglycol, 200 mM NaCl and 2.5 mM MgCl₂) and incubated at 90° C. for 15 min. Destruction of the particle conformation was analyzed by AAV2 titration ELISA recognizing a conformational epitope within the native capsid. Protein concentration of the heat-denatured particles was determined by Micro BCA assay (Pierce) and analyzed by Western blotting using a polyclonal anti-AAV2 antibody generated by immunization of rabbits with purified VP3 protein of AAV2 (data not shown).

Rabbits were immunized with heat-denatured AAV-TP11-2× particles (5.7 μg per application) or AAV-TP18-2× particles (1.8 μg per application) s.c. in the presence of an adjuvant provided by Biogenes as described above. 2 weeks after an initial prime immunization rabbits were boosted with the heat-denatured particles. Serum of the animals was analyzed 2 weeks after the boost immunization for levels of CETP auto-antibodies as described above. In a control group rabbits were vaccinated with native AAV-TP11-2× or AAV-TP18-2× particles using the same regimen as for the heat-denatured particles.

Analysis of the CETP auto-antibody titer in the sera of the immunized animals demonstrates that destruction of the native capsid conformation results in a strongly impaired induction of CETP antibodies compared with the native vaccine (FIG. 22) showing that the native capsid structure and the structured presentation of the epitopes within the capsid is essential for breaking of self-tolerance.

10.4. Evaluation of the Impact of Anti-AAV2 Antibodies on Immunization with AAV2-Based Vaccines

The immunization experiments demonstrated that AAV-based vaccines induce high titers of anti-AAV capsid antibodies in addition to the target specific antibodies (data not shown). However, most humans are AAV2 positive meaning that these people have anti-AAV2 antibody titers that potentially might affect vaccination results using AAV2-based particles. To evaluate the impact of anti-AAV2 antibodies on the immunization success of AAV2-based vaccines, rabbits were pre-immunized by two applications of wtAAV2 (4.5 μg per application), before immunization (prime and two boost immunizations) with an AAV2-based CETP vaccine (AAV-TP18) was started. wtAAV2 particles were administered s.c. or i.m. in the presence of an adjuvant provided by Biogenes as described above. 2 weeks after an initial prime immunization with wtAAV2, rabbits were boosted once again with wtAAV2. Serum was analyzed two weeks after the prime and 1^(st) boost immunization for the level of anti-AAV2 antibodies. The anti-AAV2 antibody titer was determined by ELISA using immobilized wtAAV2 particles as described below. The data demonstrate that high levels of anti-wtAAV2 antibodies are detectable after two applications of wtAAV2 for both s.c. and i.m. administration (FIG. 23A).

3 weeks after boost immunization with wtAAV2, rabbits received the first prime immunization with the AAV2-based vaccine AAV-TP18 (7.2 μg per application). The vaccine was administered s.c. or i.m. in the presence of adjuvant provided by Biogenes as described above. Rabbits were boosted with the vaccines 2 weeks after the prime vaccination. Sera were analyzed 2 weeks after the boost vaccination for the level of CETP auto-antibodies (FIG. 23B). CETP auto-antibody titers were determined as described above. Results were compared to vaccination (s.c.) of animals not pre-immunized with wtAAV2.

The data demonstrate that wtAAV2 pre-immunization results in high titers of anti-AAV2 capsid antibodies. However, these high anti-AAV2 capsid antibodies do not impair the immunization success of an AAV2-based vaccine, in this case regarding the induction of anti-CETP auto-antibodies. Accordingly, it is concluded that AAV2 sero-positive humans are equally eligible for vaccination with AAV2-particles as sero-negative humans and that sero-conversion of a vaccinated human during a vaccination protocol does not impair vaccination success.

Determination of Anti-wtAAV2 Antibody Titers:

The anti-AAV2 antibody titer was determined by ELISA using immobilized wtAAV2 particles. Briefly, 5×10⁹ wtAAV2 particles were immobilized in each well of a 96-well Maxisorp plate (Nunc) in a total volume of 50 μl PBS per well. The plate was incubated at 37° C. for 1 h. After blocking of the wells with PBS, 5% skim milk, 0.1% Tween-20, immobilized wtAAV2 particles were incubated with serial dilutions of the immune sera in dilution buffer (PBS with 1% skim milk, 1% BSA, 0.1% Tween-20) for 1 h at 37° C. Rabbit pre-immune sera or rabbit sera of unrelated vaccinations served as negative controls. After washing, binding of rabbit IgG to the immobilized AAV2 was detected using a HRP-labelled anti-rabbit IgG antibody and TMB as substrate. Antibody titers were determined as described above.

10.5. Prime/Boost Regimen for AAV-Based Vaccines

16.4 μg AAV2 particles carrying the CETP-intern epitope (CDAGSVRTNAPD, SEQ ID NO: 123) at position I-453 and I-587 (AAV2-CETin-2×) were administered i.m. at each prime or boost immunization together with the adjuvant provided by Biogenes as described above.

Three different regimens were evaluated. Group A received one prime and three boost applications of AAV2-CETin-2× (AAV2-based vaccination). Group B received one prime and one boost immunization with AAV2-CETin-2× followed by two boost immunizations with the LPH-coupled CETP-intern peptide (LPH-peptide boost). Group C received one prime and one boost immunization with AAV2-CETIn-2× followed by two boost immunizations with AAV1-CETin (AAV1 particle carrying the CETP-intern epitope at position I-588; 11.7 μg/application). In each group the first boost immunization was performed two weeks after the prime immunization. The 2^(nd) and 3^(rd) boost immunization was performed three weeks after the preceding boost vaccination.

Immune sera were analyzed for anti-CETP-reactivity (CETP auto-antibody titer) two weeks after the 1st, 2nd and 3rd boost immunization as described above (FIG. 24).

These data demonstrate that high levels of CETP auto-antibodies are detectable in animals vaccinated with AAV2-CETin-2× only (group A). There is no increase of CETP auto-antibodies observed in the group of animals boosted with LPH-coupled CETP peptide (group B). Furthermore, data demonstrate that switching of the serotype of the AAV-backbone (group C) has the potential to increase the immune response to a self-antigen compared to boost vaccinations with an individual AAV serotype.

10.6. Evaluation of the Impact of Different Adjuvants on Immunization with AAV2-Based Vaccines

Since the adjuvant provided by Biogenes may not be suitable for application in humans, alternative adjuvants were evaluated. In a first approach Montanide ISA 51 VG sterile (Seppic) was tested. Rabbits were immunized with the CETP vaccine AAV-TP18 (7.2 μg per application) i.m. or s.c. The volume of the purified vaccine was adjusted to 0.5 ml with formulation buffer and mixed with an equal volume of Montanide ISA 51 VG sterile. A control group was immunized s.c. with AAV-TP18 using the adjuvant provided by Biogenes as described above. For each vaccination approach two rabbits were immunized four times (one prime and three boost immunizations). The first boost immunization was performed 2 weeks after an initial prime immunization. Rabbits were boosted another two times with the vaccines at intervals of 3 weeks. Immune sera were analyzed for anti-CETP-reactivity (CETP auto-antibody titer) two weeks after the 1st, 2nd and 3rd boost immunization as described above. Analysis of the CETP auto-antibody titers of animals vaccinated s.c. with AAV-TP18 in the presence of Montanide ISA 51, demonstrates that similar titers are induced as in the vaccination approach using the Biogenes adjuvant (FIG. 25). Comparison of s.c. and i.m. administration of the AAV-TP18 vaccine in the presence of Montanide ISA 51 shows, that higher auto-antibody titers are generated by i.m. vaccination. These data demonstrate that AAV-base vaccines are able to induce auto-antibodies in the presence of a clinically applicable adjuvant like Montanide ISA 51 VG sterile.

In addition, the combination of AAV-based vaccine with other adjuvants such as aluminum based adjuvant Alhydrogel 2% can be evaluated with respect to to induction of auto-antibodies accordingly.

10.7. Vaccination of Rabbits with an AAV1-Based CETP Vaccine

In order to prove that results obtained with AAV2 based particles can easily be transferred to other AAV-serotypes or other parvoviruses the CETP-intern peptide (CDAGSVRTNAPD, SEQ ID NO: 123) had been inserted into the AAV1 capsid as described in 7.4.1.

For the vaccination approach two rabbits were immunized i.m. with 11.7 μg each of the construct AAV1-CETP-588 (insertion of CETP-intern epitope at position 588) as described in 10.2. The first boost immunization was performed 2 weeks after an initial prime immunization. Rabbits were boosted another 2 times with the vaccines at intervals of 3 weeks. Serum of the immunized animals was prepared two weeks after each boost immunization. CETP auto-antibody titers were determined as described above.

Data obtained demonstrate that the AAV1-based CETP vaccine AAV1-CETP-588 induces high levels of CETP auto-antibodies (FIG. 26). The induction of CETP auto-antibody was at least comparable to AAV2-CETin-2× vaccination (see 10.5). From this experiment it can be concluded that the AAV2 backbone can be substituted by the AAV1 backbone.

10.8. Vaccination Against Human β-Amyloid

For vaccination against human β-amyloid 1.0 μg of AAV2 particles carrying the human β-amyloid (aa 1-9)-epitope (DAEFRHDSG, SEQ ID NO: 158) at position I-587 were administered s.c. at each prime or boost immunization in the presence of the adjuvant provided by Biogenes. Two rabbits were immunized four times (one prime and three boost immunizations). The first boost immunization was performed 2 weeks after an initial prime immunization. Rabbits were boosted another two times with the vaccine at intervals of 3 weeks. Immune sera were analyzed for anti-β-amyloid reactivity two weeks after the 1^(st), 2^(nd) and 3^(rd) boost immunization as described below.

Resulting data demonstrate that immunization of rabbits with the AAV2-based β-amyloid vaccine efficiently induces antibodies against β-amyloid (FIG. 27).

Determination of Anti-β-Amyloid Antibody Titers:

The anti-β-amyloid antibody titer was determined by ELISA using immobilized Aβ(1-42) (Biosource) as antigen. Briefly, 250 ng Aβ(1-42) peptide was immobilized in each well of a 96-well Maxisorp plate (Nunc) in a total volume of 50 μl PBS per well. The plate was incubated at 37° C. for 1 h. After blocking of the wells with PBS/5% skim milk/0.1% Tween-20, immobilized Aβ(1-42) was incubated with serial dilutions of the immune sera in dilution buffer (PBS with 1% skim milk, 0.1% Tween-20) for 1 h at 37° C. Rabbit pre-immune sera or rabbit sera of unrelated vaccinations served as negative controls. After washing binding of rabbit IgG to immobilized Aβ(1-42) was detected using a HRP-labelled anti-rabbit IgG antibody and TMB as substrate. Antibody titers were determined as described above.

10.9. Immunization Against Human IgE Using AAV-Based Vaccines

A panel of AAV-based vaccines carrying epitopes derived from human IgE was generated as described above. AAV-based IgE vaccines were compared to the corresponding peptide vaccines containing the same epitope coupled to LPH as carrier protein. The peptides were chemically synthesized with a C- or N-terminal cystein residue that was used for coupling of the peptides to LPH. Synthesis and coupling of the peptides was performed by Biogenes (Berlin, Germany).

The following vaccines were used for immunization of rabbits:

TABLE 25 AAV- and LPH-based vaccines used for immunization against human IgE Name of Vaccine Insertion Dose vaccine carrier Site Epitope (μg) Appl. AAV-Kricek AAV2 I-587 VNLTWSRASG 3.1 s.c. (SEQ ID NO: 85) AAV-3DEpi3 AAV2 I-587 3DEpi3 4.4 s.c. AAV-Flex AAV2 I-587 Flex 16.3 i.m. AAV-Bind2 AAV2 I-587 Bind2 5.1 i.m. LPH-Kricek LPH N/A VNLTWSRASGC see text i.m. SEQ ID NO: 326 LPH-3DEpi3 LPH N/A CDSNPRGVSAYLSR see text i.m. SEQ ID NO: 327 LPH-Flex LPH N/A CEDGQVMDVDLS see text i.m. SEQ ID NO: 328 LPH-Bind2 LPH N/A CEKQRNGTLT see text i.m. SEQ ID NO: 329

For each vaccination approach two rabbits were immunized with the vaccines shown in the table above four times (one prime and three boost immunizations). The first boost immunization was performed 2 weeks after an initial prime immunization. Rabbits were boosted another two times with the vaccines at intervals of 3 weeks.

The purified AAV-based vaccines were mixed with an equal volume of formulation buffer (PBS with 1% sorbitol, 0.2% Tween-20, 25% propylenglycol, 200 mM NaCl and 2.5 mM MgCl₂) for stabilization of the particles and stored at −80° C. until administration. If necessary, the volume of the vaccine was adjusted to 0.3 ml-0.5 ml with formulation buffer directly before application. The AAV-based vaccines were administered s.c. or i.m. together with the Biogenes adjuvant (total volume 1 ml).

The LPH-coupled peptides (in 0.3 ml TBS) were administered i.m. in the presence of 0.7 ml of the adjuvant provided by Biogenes. 1 mg of the LPH-peptide conjugate was administered for the prime immunization. 0.5 mg of the conjugate was used for the 1^(st) boost immunization and 0.25 mg of the conjugate were used for the 2^(nd) and 3^(rd) boost immunization.

Induction of anti-human IgE antibodies in the vaccinated animals was determined by ELISA using human IgE (Diatec, Oslo, Norway) as antigen. A 96-well Maxisorp plate (Nunc) was coated with human IgE (1 μg/well) for 1 h at 37° C. After coating wells were washed with wash buffer (PBS/0.1% Tween-20) and subsequently incubated with blocking buffer (5% skim milk in wash buffer) for 1 h at 37° C. After blocking of the wells, immobilized human IgE was incubated with serial dilutions of the immune sera in dilution buffer (wash buffer with 1% skim milk and 1% BSA) for 1 h at 37° C. Rabbit pre-immune sera or rabbit sera of unrelated vaccinations served as negative controls. After washing binding of rabbit IgG to the immobilized IgE was detected using a HRP-labelled anti-rabbit IgG antibody (DAKO; 1:2500 in dilution buffer). Signals (OD) were detected using TMB (KemEnTec) as substrate.

In addition to the IgE titers, the anti-peptide titers of the immune sera were analyzed. The free peptides (corresponding to the epitopes integrated in the AAV capsid or coupled to LPH) were covalently immobilized in a 96-well plate (REACTI-BIND™ Amine-binding, Maleic Anhydride Activated Plates; PIERCE) as described above. After blocking of the wells, immobilized peptides were incubated with serial dilutions of the immune sera in dilution buffer (PBS with 1% skim milk, 1% BSA, 0.1% Tween-20) for 1 h at 37° C. Rabbit pre-immune sera or rabbit sera of unrelated vaccinations served as negative controls. After washing binding of rabbit IgG to the immobilized CETP was detected using a HRP-labelled anti-rabbit IgG antibody (DAKO; 1:2500 in dilution buffer). Signals (OD) were detected using TMB (KemEnTec) as substrate. Antibody titers were determined as described above

The anti-IgE titers of the immune sera are summarized in Table 26 below:

TABLE 26 Mean anti-IgE titer of immunizations with AAV- vs. LPH-based IgE vaccines anti-IgE Titer anti-IgE Titer anti-IgE Titer Vaccine 1^(st) Boost 2^(nd) Boost 3^(rd) Boost AAV-Kricek 4750 20150 25460 AAV-Kricek* n.d. 7950 27000 AAV-3DEpi3* 5000 18200 30140 AAV-Bind2 575 3075 7750 AAV-Flex 17200 40300 38100 LPH-Kricek n.d. 1300 400 LPH-3DEpi3 705 1400 1600 LPH-Flex 15000 14000 23250 LPH-Bind2 0 0 0 *AAV-based vaccines were used for the prime and 1^(st) boost immunization; 2^(nd) and 3^(rd) boost immunization were performed with the corresponding LPH-coupled peptide

Interestingly, vaccination of rabbits with LPH-Kricek, LPH-3DEpi3 or LPH-Bind2 failed to induce significant levels of antibodies against human IgE. The immunogenic properties of the peptide based vaccines are reflected by the high titers of peptide specific antibodies induced by the peptide vaccines (data not shown). However, these antibodies show no or only weak reaction with native human IgE. Only LPH-Flex induced reasonably high titers of antibodies specific for native human IgE. This is in clear contrast to the results obtained with the corresponding AAV-based vaccines like AAV-Kricek (FIG. 28) which generate considerably higher human IgE specific antibody titers compared to the corresponding LPH-fusion constructs. This indicates that the fixed conformation of the corresponding IgE epitopes in the AAV2 capsid resembles the structure of the sequence within the IgE molecule in a better way than the LPH-coupled peptides. It should be noted that the generation of anti-human IgE antibodies in this animal model with rabbits does not overcome tolerance of the immune system to self-antigens.

For evaluation of the safety and efficacy of the AAV-based anti-human IgE vaccines in non-human primate models (e.g. cynomolgus monkeys) it is critical that the human and non-human primate IgE epitope is identical in both species. The cynomolgus IgE sequence (Fc region) was sequenced at the German Primate Centre (Gottingen, Germany). The sequence of cynomolgus IgE is shown below. Sequence alignment of human and cynomolgus IgE (data not shown) revealed that the Kricek and 3DEpi3 epitope are identical in both species. Therefore, cynomologus is a suitable animal model for safety and efficacy testing of vaccines carrying the Kricek or 3DEpi3 epitope. From sequencing data it is not fully clear whether at position 86 there is an M(bold) or T. This may be due to a sequencening error or a polymorphism. The human sequence has an M at this position.

Sequence of cynomolgus IgE (Fc region): (SEQ ID NO: 233) 001 SVFTASIQSP FVFPLIPCCK HIASNATSVT LGCLATGYFP EPVMVTWDAG 051 SLNRSTMTLP ATTFTPSGHY ATISLLTVSG AWAKEMFTCH VVHTPSSADK 101 EVNKTFGVCS RNFTPPTVKI LQSSCDDDGH FPPTIQLLCL ISGYTPGAIN 151 VTWLENGQVM KVNSPTPPAT QEGELASTQS EFTLAQKHWL SDRTYTCQVT 201 YQGTTYNDST KKCADSNPRG VSAYLSRPSP FDLFISKSPT ITCLVVDLAP 251 SKETVNLTWS RASGKPVPHI PATEKKQQRN GTLTVTSILP VVTQDWIEGE 301 TYQCRVTHPH LPRALVRSMT KTSGPRAAPE VYVFATPEKL ESRDKRTLAC 351 LIQNFMPEDI SVQWLHSDVQ LPDARHSVTQ PRKTKGSGFF VFSRLEVTKA 401 EWEQKDEFIC RAVHEAASPS WIVQQAVSVN PGK* 10.10. Vaccination of Rabbits with an IgE Epitope Fused to a Synthetic T-Helper Epitope

According to published data ({Wang, 2003 #65}; WO 99/67293), the IgE derived peptide CGETYQSRVTHPHLPRALMRSTTKC (SEQ ID NO: 234) is able to induce high titers of neutralizing anti-IgE antibodies in mice if a disulfide bond is formed between the terminal cystein terminal residues (shown in bold printed letters) and the cyclic peptide is linked to a synthetic T-helper epitope according to the following scheme:

(SEQ ID NO: 235) TAKSKKFPSYTATYQFGGKKKIITITRIITIITTIDGGC*GETYQSRVTH PHLPRALMRSTTKC* *linked by a disulfide bond

To evaluate this IgE peptide vaccine in our immunization experiments, the vaccine (“Wang peptide”) was synthesized (Activotec) and used for immunization of rabbits (Biogenes). 100 μg of the peptide vaccine (in 0.2 ml PBS) was administered i.m. in the presence of the adjuvant provided by Biogenes.

In addition to the complete IgE derived sequence described by Wang et al. a shortened sequence of this peptide containing a putative B-cell epitope called “Wang-CS” was synthesized, coupled to LPH (via an additional N-terminal cystein residue) and used for i.m. vaccination of rabbits in the presence of the adjuvant provided by Biogenes. The LPH-coupled peptide Wang-CS (in 0.3 ml TBS) were administered i.m. in the presence of 0.7 ml of the adjuvant provided by Biogenes. 1 mg of the LPH-peptide conjugate was administered for the prime immunization. 0.5 mg of the conjugate was used for the 1^(5t) boost immunization and 0.25 mg of the conjugate were used for the 2^(nd) and 3^(rd) boost immunization.

Rabbits were immunized with the different peptide vaccines four times (one prime and three boost immunizations). The first boost immunization was performed 2 weeks after an initial prime immunization. Rabbits were boosted another two times with the vaccines at intervals of 3 weeks. Immune sera were analyzed for anti-human IgE reactivity two weeks after the 1st, 2nd and 3rd boost immunization as described above.

These data demonstrate that vaccination of rabbits with the short LPH-coupled peptide “Wang-CS” results in anti-IgE titers that are in the range of the titers obtained with the vaccine “Wang peptide” described above (FIG. 29).

Since published data demonstrate that the Wang-peptide induces neutralizing anti-IgE antibodies in vaccinated animals (WO 99/67293), the functional properties of these polyclonal antibodies was evaluated in a cellular histamine release assay (see below).

The “Wang-CS” sequence was inserted into the AAV2 capsid at position I-587 as described above and will be used for vaccination experiments. A major advantage of the AAV-based vaccines carrying the epitope “Wang-CS” or the full-length sequences GETYQSRVTHPHLPRALMRSTTK (SEQ ID NO: 236) or “Wang” GETYQCRVTHPHLPRALMRSTTK (SEQ ID NO: 212) is their high potential of breaking self-tolerance and induction of high levels of anti-human IgE auto-antibodies.

11. Characterization of Anti-Human IqE Antibodies in Cellular Assays

11.1. Purification of Total IgG from Serum of Vaccinated Rabbits

Total IgG of the immune sera was prepared using a commercially available kit (Proteus) based on the interaction of rabbit IgG with protein A. Purification was performed according to the protocol provided by the manufacturer. Protein concentration of total IgG was analyzed by MICRO BCA™ protein assay (PIERCE); purity of the prepared total IgG was analyzed by SDS-PAGE and colloidal Coomassie staining (data not shown).

11.2. Evaluation of the Anaphylactic Properties of the Anti-IgE Antibodies

To evaluate whether the polyclonal anti-IgE antibodies induced by vaccination of rabbits are anaphylactic, the effect of the anti-IgE antibodies on IgE mediated degranulation of basophils was investigated (Takagi et al., 2003). Rat basophilic RBL2H3 cells (1×10⁵ cells) overexpressing the alpha-chain of human FcεRI were sensitized by incubation with 250 ng/ml human IgE (Dianova) for 2 h in a total volume of 200 μl RPMI medium (supplemented with 10% FCS and NEAA) in a 96-well plate. Cells were washed with medium and resuspended in 100 μl Tyrode's salt solution (Sigma) supplemented with 0.1% BSA. Polyclonal anti-IgE antibodies (total IgG fraction of immunized rabbits) were added to the sensitized cells at a maximum concentration of 3 mg/ml total IgG. Different concentrations of the anaphylactic monoclonal anti-IgE antibody Le27 were used as positive control. Rabbit total IgG derived from unrelated immunizations (i.e. vaccinations against CETP or β-amyloid) was used as negative control. Cells were incubated for 1 h and histamine release was measured using a commercially available histamine ELISA (Neogen).

Resulting data demonstrate that none of the evaluated polyclonal anti-human IgE antibodies induced by vaccination of rabbits with AAV-based IgE vaccines (AAV-Kricek, AAV-3DEpi3 or AAV-Flex) induces the degranulation of IgE sensitized basophils demonstrating that these anti-IgE antibodies have no detectable anaphylactic properties (FIG. 30).

11.3. Evaluation of the IgE Neutralizing Properties of the Anti-IgE Antibodies

To evaluate whether the polyclonal anti-IgE antibodies induced by vaccination of rabbits are able to neutralize IgE, the effect of the anti-IgE antibodies on IgE mediated degranulation of basophils was investigated. Human IgE (250 ng/ml; Dianova) was pre-incubated with the polyclonal anti-IgE antibodies (3 mg/ml total IgG fraction) for 2 h at RT. As a positive control human IgE was pre-incubated with XOLAIR® (1 μg/ml). Rat basophilic RBL2H3 cells (1E+05 cells) overexpressing the alpha-chain of human FcεRI were sensitized by incubation with the human IgE/anti-IgE complexes for 2 h in a total volume of 100 μl RPMI medium (supplemented with 10% FCS and NEAA) in a 96-well plate. Cells were washed once with medium and once with Tyrode's salt solution and were subsequently resuspended in 100 μl Tyrode's salt solution (Sigma) supplemented with 0.1% BSA. The anaphylactic monoclonal anti-IgE antibody Le27 (100 nM) was used for cross-linking of receptor bound IgE. Cells were incubated for 1 h with Le27 and histamine release was measured using a commercially available histamine ELISA (Neogen).

Data obtained demonstrate that the polyclonal anti-IgE antibodies induced by vaccination of rabbits with AAV-Kricek or AAV-3DEpi3 reduce the IgE mediated histamine release by about 30% (FIG. 31) The polyclonal anti-IgE antibodies induced by vaccination of rabbits with the “Wang-peptide” (see above) inhibit the histamine release by about 20%. No significant effect was observed for polyclonal antibodies obtained from immunization of rabbits with AAV-Flex or unrelated vaccinations (i.e. vaccinations against CETP or β-amyloid).

12. Evaluation of Additional Epi- or Mimotope Insertion Sites within the AAV2-Backbone

Two different strategies were followed for introduction of integration sites within the AAV2 capsid

-   a) Insertion of foreign epitopes at a defined insertion site (e.g.     I-328) -   b) Insertion by deletion of amino acid residues of AAV2 capsid and     substitution by a given epi- or mimotope sequences (e.g. Δ324-332)

TABLE 27 Insertion sites within the AAV2 capsid Integration Site AAV2 sequence at integration site I-261 YKQIS₂₆₁ SQSGA SEQ ID NO: 24 I-328 TQNDG₃₂₈ TTTIA SEQ ID NO: 330 Δ324-332 KEVTQNDGTTTIANN SEQ ID NO: 331 Δ374-380 MVPQYGYLTLNNGS SEQ ID NO: 332 Δ566-575 EEEIRTTNPVATEQYGS SEQ ID NO: 333 I-534 EEKFF₅₃₄ PQSGV SEQ ID NO: 31 I-573 NPVAT₅₇₃ EQYGS SEQ ID NO: 32 I-709 NKSVN₇₀₉ VDFTV SEQ ID NO: 334 Δ708-714 SNYNKSVNVDFTVDTNG SEQ ID NO: 335 Insertion sites are marked with reference to the preceding amino acid; Deleted/substituted sequences are depicted in bold letters.

For insertion of epi- or mimotope sequences into sites as listed in Table 27 two restriction sites (MroI/AscI) were inserted into the vector pCR-Kotin-C11 at the positions shown in the table above. The vector pCR-Kotin-C11 contains the complete AAV2 genome without ITRs and contains the following substitutions of amino acids within the cap gene: R459K, Y500F, G512D, N551D, A664T ((Endell, 2006 #711), page 45).

Insertion sites were introduced by site directed mutagenesis using the QICKCHANGE® II Site directed Mutagenesis kit (STRATEGENE) together with the primers listed in Table 28.

TABLE 28 Primers used for site directed mutagenesis of AAV2 Cap Insertion Mutagenesis primer 1 Site (universe) Mutagenesis primer 2 (reverse) I-261 5′-c tac aaa caa att tcc 5′-ggc tcc tga ttg gct TCC GGC GCG CCA GGA TCC GGA GGA TCC TGG CGC GCC gga aat agc caa tca gga gcc-3′ ttg ttt gta g SEQ ID NO: 336 SEQ ID NO: 337 I-328 5′-gtc acg cag aat gac 5′-ggc aat cgt cgt cgt TCC ggt GGC GCG CCA GGA TCC GGA TCC TGG CGC GCC acc gtc GGA acg acg acg att gcc- att ctg cgt gac 3′ SEQ ID NO: 339 SEQ ID NO: 338 I-534 5′-c gat gaa gaa aag ttt 5′-gag aac ccc gct ctg agg ttt GGC GCG CCA GGA TCC TCC GGA TCC TGG CGC GCC aaa GGA cct cag agc ggg gtt aaa ctt ttc ttc atc g-3′ ctc-3′ SEQ ID NO: 341 SEQ ID NO: 340 I-573 5′-cc aat ccc gtg gct acg 5′-ga tac aga acc ata ctg GGC GCG CCA GGA TCC GGA ctc TCC GGA TCC TGG CGC GCC gag cag tat ggt tct gta cgt agc cac ggg att gg-3′ tc-3′ SEQ ID NO: 343 SEQ ID NO: 342 I-709 5′-ctac aac aag tct gtt 5′-c cac agt aaa gtc cac TCC aat GGC GCG CCA GGA TCC GGA TCC TGG CGC GCC att aac GGA gtg gac ttt act gtg aga ctt gtt gta g-3′ g-3′ SEQ ID NO: 345 SEQ ID NO: 344 Δ 374-380 5′-gac gtc ttc atg gtg 5′-gc ctg act ccc gtt gtt cca GGC GCG CCA GGA TCC TCC GGA TCC TGG CGC GCC tgg GGA aac aac ggg agt cag cac cat gaa gac gtc-3′ gc-3′ SEQ ID NO: 347 SEQ ID NO: 346 Δ 324-332 5′-c att caa gtc aaa gag 5′-gct ggt aag gtt att ggc gtc GGC GCG CCA GGA TCC TCC GGA TCC TGG CGC GCC gac GGA gcc aat aac ctt acc ctc ttt gac ttg aat g-3′ agc-3′ SEQ ID NO: 349 SEQ ID NO: 348 Δ 566-575 5′-ca gac gaa gag gaa atc 5′-ggt aga tac aga acc ata GGC GCG CCA GGA TCC GGA TCC GGA TCC TGG CGC GCC gat tat ggt tct gta tct acc- ttc ctc ttc gtc tg-3′ 3′ SEQ ID NO: 351 SEQ ID NO: 350 Δ 708-714 5′-cc aac tac aac aag tct 5′-ga ata cac gcc att agt GGC GCG CCA GGA TCC GGA gtc TCC GGA TCC TGG CGC GCC gac act aat ggc gtg tat aga ctt gtt gta gtt gg-3′ tc-3′ SEQ ID NO: 353 SEQ ID NO: 352

Introduction of the AscI/MroI restriction site also resulted in the insertion of a new BamHI restriction site located between the AscI/MroI site. Deletion of a given sequence was also performed by site directed mutagenesis using the primers shown in Table 28. Deletion of the sequences using these primers results in the insertion of a MroI and AscI restriction site at the corresponding positions.

The EcoNI/SnaBI restriction fragments of pRC-Kotin C11 containing the new insertion sites were sub-cloned into the vector pUCAV2 for production of AAV-particles.

To evaluate whether an epi- or mimotope can be integrated at the newly created insertion sites a CETP epitope (CETP-intern) was inserted at the corresponding positions. The CETP epitope was cloned into the new AscI/MroI restriction site of pUCAV2 using annealed oligonucleotides CETin-AscI-uni and CETin-MroI-rev:

CETin-AscI-uni (SEQ ID NO: 354) 5′-CGCG GGC GGA tgc gac gcc ggc agt gtg cgc acc aat gca cca gac GGT GGC G-3′ CETin-MroI-rev (SEQ ID NO: 355) 5′-CCGG C GCC ACC gtc tgg tgc att ggt gcg cac act gcc ggc gtc gca TCC GCC-3′

Annealing of the universe and reverse oligonucleotide results in a dsDNA fragment with 5′ and 3′ site overhangs (shown in upper case letters) complementary to MroI and AscI restricted pUCAV2. The annealed oligonucleotides encode the CETP-intern epitope sequence (encoded by the oligonucleotide sequence shown in lower case letters) flanked by alanine/glycine residues. The annealed were cloned into the AscI/MroI restriction site of the modified pUCAV2 as described above. The epitope (shown in bold printed letters) is flanked by an alanine/glycine linker within the AAV capsid according to the following scheme:

GAGG CDAGSVRTNAPD GGAG (SEQ ID NO: 356)

The AAV variants were produced in small-scale as described above and the capsid titer of the cell lysate was measured using a commercially available AAV2 ELISA (Progen) based on the A20 mAb recognizing a conformational epitope within the AAV2 capsid (A20 ELISA). To quantify AAV2 variants with a modified capsid conformation that are not recognized by the AAV2 ELISA (A20 negative particles), capsids were produced in large-scale, purified by iodixanol gradient centrifugation as described above and quantified using an ELISA based on the mAb B1 (Progen). B1 mAb recognizes a linear epitope sequence at the C-terminus of the capsid proteins that is not modified by the insertion of the epitope. For quantification of AAV variants, the purified particles found in the 40% phase of the iodixanol gradient were denatured by heat-treatment, immobilized on a MaxiSorp 96-well plate (Nunc) and detected by the B1 mAb followed by a HRP-conjugated anti-mouse IgG antibody. In parallel, a standard curve was generated by immobilizing a dilution series of heat-denatured wtAAV2 capsids with a known capsid titer. The standard curve was used for quantification of the AAV variants.

The data of the B1 and A20 based ELISAs demonstrate that insertion of the CETP epitope at positions I-534 or I-573 as well as I-261 results in formation of particles that are recognized by B1 but not by A20 ELISA (Table 29). Particles with the CETP epitope at position I-328 can hardly be detected within the 40% phase of the iodixanol gradient by A20 or B1 ELISA. The difference between the capsid titers of the variant Δ566-575 in the 40% iodixanol phase measured by A20 or B1 ELISA is likely due to the deletion of a known minor A20 epitope {Wobus, 2000 #67} that results in a lower affinity of this variant to A20 mAb in the A20-based titration ELISA (Table 29).

No particle formation was observed for the variants with the CETP epitope integrated at position Δ324-332, Δ374-380, Δ708-714 or I-709.

TABLE 29 Capsid titers (capsids/ml) of AAV2-variants carrying the CETP-intern epitope Insertion Site A20 ELISA B1 ELISA I-328 5.2 × 10¹⁰ BDL I-261 BDL 1.1 × 10¹³ I-573 BDL 1.1 × 10¹³ I-534 BDL 2.6 × 10¹³ Δ566-575 1.5 × 10¹² 1.4 × 10¹³ BDL: below detection limit of the ELISA

To evaluate whether the CETP epitope is located at the capsid surface of the new variants, the purified particles (40% iodixanol phase) were dotted onto a membrane (5.0×10¹¹ or 1.0×10¹¹ particles per dot). As a positive control AAV2 particles carrying the CETP-intern epitope at position I-453 and I-587 (AAV2-CETin-2×) were dotted. As a negative control, an AAV2 variant carrying an unrelated CETP epitope (TP10) was dotted. The blot was incubated with a polyclonal immune serum directed against the CETP-intern epitope that was generated by immunization of rabbits with the LPH-coupled CETP-intern peptide. Binding of the CETP antibody to the AAV-variants was detected using an HRP-conjugated anti-rabbit IgG antibody (FIG. 32).

The data demonstrate that for the new capsid variants Δ566-575 (I-570), I-534, I-573, I-261 and I-328 the CETP epitope is recognized by the antibody proving that the epitope is located at the surface of the capsids. There is no unspecific cross-reaction of the CETP antibody with the AAV-capsid, since the AAV variant AAV-TP10 is not recognized by the antibody. Accordingly I-261, I-573, I-534 and insertion by substitution Δ566-575 are further preferred insertion sites regarding all aspects of the present invention.

Corresponding insertion sites of different AAV serotypes or different parvoviruses can be taken from FIG. 1 as depicted for I-453 and I-587.

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1. A method for identifying a parvovirus mutated structural protein capable of specifically binding to a binder for an antigen, the method comprising the steps of: a) providing a library of parvovirus virions expressing at least one mutated parvovirus structural protein, b) providing a binder for an antigen, c) selecting at least one parvovirus virion specifically binding to the binder, and d) identifying i) the parvovirus mutated structural protein or a mutated part thereof, or ii) the gene or a mutated part thereof encoding the parvovirus mutated structural protein of the parvovirus virion selected in step c).
 2. The method of claim 1 wherein the at least one parvovirus virion selected in step c) is amplified by viral replication and subsequent packaging in a production cell under suitable conditions, and wherein at least steps b) to c) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.
 3. The method of claim 1, wherein the selecting step is performed using a binder immobilized on a carrier.
 4. The method of claim 1, wherein the selecting step is performed using a binder in suspension.
 5. The method of claim 1, wherein said selecting at least one parvovirus virion further comprises selecting for non-binding to a second binder.
 6. The method of claim 1, wherein said method further comprises the steps of e) randomizing the gene encoding the parvovirus mutated structural protein, f) packaging the randomized genes into a further library of parvoviruses, and g) repeating the steps a)-d).
 7. The method of claim 1, wherein the parvovirus mutated structural protein further comprises at least one random mutation compared to the respective parvovirus wild-type structural protein.
 8. The method of claim 7, wherein the parvovirus is selected from the group consisting of adeno-associated virus (AAV), bovine AAV (b-AAV), canine AAV (CAAV), canine parvovirus (CPV), mouse parvovirus, minute virus of mice (MVM), B19, H1, avian AAV (AAAV), feline panleukopenia virus (FPV), and goose parvovirus (GPV).
 9. The method of claim 8, wherein the AAV is AAV-1, AAV-2, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11 or AAV-12.
 10. The method of claim 1, wherein the library has a multiplicity of parvoviral mutants of greater than 10⁵.
 11. The method of claim 1, wherein the parvovirus mutant structural protein comprises at least one insertion of 4-30 amino acids.
 12. The method of claim 11, wherein said insertion is of 5-20 amino acids.
 13. The method of claim 12, wherein said insertion is 5-15 amino acids.
 14. The method of claim 11, wherein the insertion comprises two cysteines capable of forming a disulfide bond to form a loop consisting of the inserted amino acids.
 15. The method of claim 11, wherein a) the insertion is inserted into one or more positions selected from the group consisting of I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-453, I-459, I-471, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, and I-716; or b) the insertion is inserted into two positions selected from the group consisting of I-261, I-453, I-534, I-570, I-573, and I-587.
 16. The method of claim 15, wherein the insertion is inserted at position I-261, I-453, I-534, I-570, I-573, or I-587.
 17. The method of claim 15, wherein said two positions are I-261 in combination with I-587 or I-261 in combination with I-453.
 18. The method of claim 1, wherein the parvovirus mutated structural protein comprises at least one further mutation selected from the group consisting of a point mutation, an internal or terminal deletion, a second insertion, and a substitution.
 19. The method of claim 18, wherein said further mutation is a second insertion and said second insertion is internal or a N- or C-terminal fusion, and has a length of 4 to 40, 5 to 30, or 7 to 20 amino acids.
 20. The method of claim 19, wherein the second insertion is a tag useful for binding to a ligand.
 21. The method of claim 1, wherein said parvovirus mutated structural protein comprises at least one B-cell epitope heterologous to the parvovirus, wherein the B-cell epitope is located on the surface of the virus.
 22. The method of claim 21, wherein said B-cell epitope is a tolerogen-derived epitope. 